Amnion derived adherent cells

ABSTRACT

Provided herein are novel angiogenic cells from amnion, referred to as amnion derived adherent cells, and populations of, and compositions comprising, such cells. Further provided herein are methods of obtaining such cells and methods of using the cells in the treatment of individuals.

This application claims benefit of U.S. Provisional Patent ApplicationNo. 61/116,248, filed Nov. 19, 2008, the disclosure of which isincorporated by reference in its entirety.

1. FIELD

Provided herein are novel angiogenic cells from amnion, and populationsof such cells, referred to herein as “amnion derived adherent cells”(AMDACs). Amnion derived adherent cells are distinct frompreviously-described placental stem cells, including tissue cultureplastic adherent placental stem cells.

2. BACKGROUND

Cell compositions, e.g., stem cell compositions, have become anattractive therapy for a number of physiological deficiencies, e.g.,bone marrow replacement. A need exists for additional populations ofcells, e.g., stem cells or progenitor cells that have angiogenicpotential and/or properties.

3. SUMMARY

In one aspect, provided herein is an isolated amnion derived adherentcell, also referred to herein as an AMDAC, wherein said cell is adherentto tissue culture plastic, and wherein said cell is OCT-4⁻ (negative forOCT-4, also known as POU5F1 or octamer binding protein 4), or asdetermined by reverse transcriptase-polymerase chain reaction (RT-PCR),e.g., as compared to an appropriate control cell line, such as anembryonal carcinoma-derived stem cell line (e.g., NTERA-2, e.g.,available from the American Type Culture Collection, ATCC NumberCRL-1973), as determined by RT-PCR for 30 cycles. In a specificembodiment, the cells are OCT-4⁻, as determined by RT-PCR, andVEGFR1/Flt-1⁺ (vascular endothelial growth factor receptor 1) and/orVEGFR2/KDR⁺ (vascular endothelial growth factor receptor 2, also knownas kinase insert domain receptor), as determined by immunolocalization.In another specific embodiment, the cells are OCT-4⁻, as determined byRT-PCR, and CD49f⁺ (integrin-α6⁺), as determined by immunolocalization.In a specific embodiment, said cell is OCT-4⁻, as determined by RT-PCR,and HLA-G⁻, as determined by RT-PCR. In another specific embodiment,said cell is OCT-4⁻, as determined by RT-PCR, and CD90⁺, CD105⁺, orCD117⁻ as determined by immunolocalization. In a more specificembodiment, said OCT-4⁻ cell is CD90⁺, CD105⁺, and CD117⁻. In anotherspecific embodiment, the cell is OCT-4⁻, and does not express SOX2,e.g., as determined by RT-PCR for 30 cycles.

In another embodiment, said OCT-4⁻ cell is one or more of CD29⁺, CD73⁺,ABC-p⁺, and CD38⁻, as determined by immunolocalization.

In another specific embodiment, said OCT-4⁻ cell is additionally one ormore of CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, Tie-2⁺ (angiopoietinreceptor), TEM-7⁺ (tumor endothelial marker 7), CD31⁻, CD34⁻, CD45⁻,CD133⁻, CD143⁻ (angiotensin-I-converting enzyme, ACE), CD146⁻ (melanomacell adhesion molecule), CXCR4⁻ (chemokine (C-X-C motif) receptor 4) asdetermined by immunolocalization. In a more specific embodiment, saidcell is CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻,CD45⁻, CD133⁻, CD143⁻, CD146⁻, and CXCR4⁻ as determined byimmunolocalization. In another more specific embodiment, the amnionderived adherent cell provided herein is OCT-4⁻, as determined byRT-PCR; VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺, as determined byimmunolocalization; and one or more, or all, of CD31⁻, CD34⁻, CD45⁻,CD133⁻, and/or Tie-2⁻ as determined by immunolocalization. In a specificembodiment, the amnion derived adherent cell, or a population of amnionderived adherent cells, expresses at least 2 log less PCR-amplified mRNAfor OCT-4 at, e.g., >20 cycles, than an NTERA-2 cell, or population ofNTERA-2 cells having an equivalent number of cells. In another specificembodiment, said OCT-4⁻ cell is additionally VE-cadherin⁻ (CD144⁻) asdetermined by immunolocalization. In another specific embodiment, saidOCT-4⁻ cell is additionally positive for CD105⁺ and CD200⁺ as determinedby immunolocalization. In another specific embodiment, said OCT-4⁻ celldoes not express CD34 as detected by immunolocalization after exposureto 1 to 100 ng/mL VEGF (vascular endothelial growth factor) for 4 to 21days.

In another aspect, provided herein is an isolated amnion derivedadherent cell, wherein said cell is adherent to tissue culture plastic,and wherein said cell is OCT-4⁻ and SOX-2⁻, as determined by RT-PCR; andCD90⁺, CD105⁺, and CD117⁻, as determined by flow cytometry. In aspecific embodiments, the OCT-4⁻, SOX-2⁻ cell is additionally HLA-G⁻ orCD271⁻, as determined by flow cytometry. In a more specific embodiment,said cell is OCT-4⁻ and SOX-2⁻, as determined by RT-PCR; and CD90⁺,CD105⁺, CD117⁻, CD271⁻ and HLA-G⁻, as determined by flow cytometry.

In another aspect, provided herein is an isolated amnion derivedadherent cell, wherein said cell is adherent to tissue culture plastic,and wherein said cell is positive for CD309 (also known as VEGFR2/KDR⁺).

In another aspect, provided herein is an isolated amnion derivedadherent cell, wherein said cell is adherent to tissue culture plastic,and wherein said cell is OCT-4⁻, as determined by RT-PCR, and one ormore of VEGFR2/KDR⁺, CD9⁺, CD54⁺, CD105⁺, CD200⁺, or VE-cadherin⁻, asdetermined by immunolocalization. In a specific embodiment, said cell isOCT-4⁻, as determined by RT-PCR at, e.g., >20 cycles, and VEGFR2/KDR⁺,CD9⁺, CD54⁺, CD105⁺, CD200⁺, and VE-cadherin⁻, as determined byimmunolocalization. In another specific embodiment, the cell does notexpress CD34, as detected by immunolocalization, after exposure to 1 to100 ng/mL VEGF for 4 to 21 days.

In another embodiment, the amnion derived adherent cell is OCT-4⁻,CD49f⁻, HLA-G⁻, CD90⁺, CD105⁺, and CD117⁻. In a more specificembodiment, said cell is one or more of CD9⁺, CD10⁺, CD44⁺, CD54⁺,CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻(melanoma cell adhesion molecule), or CXCR4⁻, as determined byimmunolocalization or flow cytometry. In a more specific embodiment,said cell is CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻,CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻, and CXCR4⁻ as determined byimmunolocalization. In another specific embodiment, said cell isadditionally VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺, as determined byimmunolocalization; and one or more of CD31⁻, CD34⁻, CD45⁻, CD133⁻,and/or Tie-2⁻ as determined by immunolocalization. In another specificembodiment, said cell is additionally VEGFR1/Flt-1⁺, VEGFR2/KDR⁺, CD31⁻,CD34⁻, CD45⁻, CD133⁻, and Tie-2⁻ as determined by immunolocalization.

In another embodiment, provided herein is an isolated amnion derivedadherent cell, wherein said cell does not express mRNA for FGF4, IFNG,CXCL10, ANGPT4, ANGPTL3, FGA, LEP, PRL, PROK1, TNMD, FLT3, XLKD1, CDH5,LECT1, PLG, TERT, SOX2, NANOG, MMP-13, DLX5, or BGLAP, as determined byRT-PCR, e.g., for 30 cycles. In another embodiment, provided herein isan isolated amnion derived adherent cell, wherein said cell does notconstitutively express one or more of invariant chain, HLA-DR-DP-DQ,CD6, CD271, as determined by flow cytometry.

Further provided herein is an isolated population of cells comprising anamnion derived adherent cell. In a specific embodiment, at least about50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of cells in said population areamnion derived adherent cells. In one embodiment, said cell is OCT-4⁻,as determined by RT-PCR, and VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺ asdetermined by immunolocalization, and wherein said isolated populationof cells is not an amnion. In another embodiment, provided herein is anisolated population of cells comprising an amnion derived adherent cellthat is OCT-4⁻ and HLA-G⁻, as determined by RT-PCR, and VEGFR1/Flt-1⁺ orVEGFR2/KDR⁺ as determined by immunolocalization; and wherein saidisolated population of cells is not an amnion. In a specific embodiment,at least about 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of cells in saidpopulation are said amnion derived adherent cells. In anotherembodiment, provided herein is an isolated population of cellscomprising an amnion derived adherent cell, wherein said cell isadherent to tissue culture plastic, wherein said cell is OCT-4⁻, asdetermined by RT-PCR, VEGFR1/Flt-1⁺ and VEGFR2/KDR⁺ as determined byimmunolocalization, wherein said cell is additionally one or more ofCD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻, CD45⁻,CD133⁻, CD143⁻, CD146⁻, or CXCR4⁻, as determined by immunolocalization,or HLA-G⁻ as determined by RT-PCR for, e.g., >20 cycles, and whereinsaid isolated population of cells is not an amnion. In a specificembodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% ofcells in said population are said amnion derived adherent cells. Inanother embodiment, provided herein is an isolated population of cellscomprising an amnion derived adherent cell, wherein said cell isadherent to tissue culture plastic, wherein said cell is OCT-4⁻, asdetermined by RT-PCR, VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺ as determined byimmunolocalization, wherein said cell does not express CD34 as detectedby immunolocalization after exposure to 1 to 100 ng/mL VEGF for 4 to 21days, and wherein said isolated population of cells is not an amnion. Ina specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%or 99% of cells in said population are said amnion derived adherentcells. In another embodiment, any of the above populations of cellscomprising amnion derived adherent cells forms sprouts or tube-likestructures when cultured in the presence of angiogenic factors such asvascular endothelial growth factor (VEGF), epithelial growth factor(EGF), platelet derived growth factor (PDGF) or basic fibroblast growthfactor (bFGF), e.g., on a substrate such as MATRIGELTm.

In another embodiment, provided herein is a population of cells, whereinat least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells in saidisolated population of cells are amnion derived adherent cells that areOCT-4⁻, as determined by RT-PCR, and positive for VEGFR2/KDR, CD9, CD54,CD105, or CD200. In a specific embodiment, at least about 50%, 60%, 70%,80%, 90%, 95% or 98% of cells in said isolated population of cells areamnion derived adherent cells that are OCT-4⁻, as determined by RT-PCR,and VEGFR2/KDR⁺, CD9⁺, CD54⁺, CD105⁺, and CD200⁺, as determined byimmunolocalization. In a more specific embodiment, said amnion derivedadherent cells do not express CD34, as detected by immunolocalization,after exposure to 1 to 100 ng/mL VEGF for 4 to 21 days. In a specificembodiment, said amnion derived adherent cells are adherent to tissueculture plastic. In another specific embodiment, said population ofcells forms sprouts or tube-like structures when cultured in thepresence of an angiogenic factor such as vascular endothelial growthfactor (VEGF), epithelial growth factor (EGF), platelet derived growthfactor (PDGF) or basic fibroblast growth factor (bFGF), e.g., on asubstrate such as MATRIGELTm.

In another embodiment, provided herein is a population of cells, e.g.,human cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98%of cells in said isolated population of cells are amnion derivedadherent cells that express RNA for one or more, or all, of ACTA2,ADAMTS1, AMOT, ANG, ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL4, BAH,CD44, CD200, CEACAM1, CHGA, COL15A1, COL18A1, COL4A1, COL4A2, COL4A3,CSF3, CTGF, CXCL12, CXCL2, DNMT3B, ECGF1, EDG1, EDIL3, ENPP2, EPHB2,FBLN5, F2, FGF1, FGF2, FIGF, FLT4, FN1, FST, FOXC2, GRN, HGF, HEY1,HSPG2, IFNB1, IL8, IL12A, ITGA4, ITGAV, ITGB3, MDK, MMP2, MYOZ2, NRP1,NRP2, PDGFB, PDGFRA, PDGFRB, PECAM1, PF4, PGK1, PROX1, PTN, SEMA3F,SERPINB5, SERPINC1, SERPINF1, TIMP2, TIMP3, TGFA, TGFB1, THBS1, THBS2,TIE1, TIE2/TEK, TNF, TNNI1, TNFSF15, VASH1, VEGF, VEGFB, VEGFC,VEGFR1/FLT1, or VEGFR2/KDR.

In another embodiment, provided herein is a population of cells, e.g., apopulation of amnion derived adherent cells, or a population of cellswherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells insaid isolated population of cells are amnion derived adherent cells thatexpress one or more of, or all of, CD49d, Connexin-43, HLA-ABC, Beta2-microglobulin, CD349, CD318, PDL1, CD106, Galectin-1, ADAM 17precursor (A disintegrin and metalloproteinase domain 17) (TNF-alphaconverting enzyme) (TNF-alpha convertase), Angiotensinogen precursor,Filamin A (Alpha-filamin) (Filamin 1) (Endothelial actin-bindingprotein) (ABP-280) (Nonmuscle filamin), Alpha-actinin 1 (Alpha-actinincytoskeletal isoform) (Non-muscle alpha-actinin 1) (F-actin crosslinking protein), Low-density lipoprotein receptor-related protein 2precursor (Megalin) (Glycoprotein 330) (gp330), Macrophage scavengerreceptor types I and II (Macrophage acetylated LDL receptor I and II),Activin receptor type IIB precursor (ACTR-IIB), Wnt-9 protein, Glialfibrillary acidic protein, astrocyte (GFAP), Myosin-binding protein C,cardiac-type (Cardiac MyBP-C) (C-protein, cardiac muscle isoform), orMyosin heavy chain, nonmuscle type A (Cellular myosin heavy chain, typeA) (Nonmuscle myosin heavy chain-A) (NMMHC-A).

In another aspect, provided herein is a population of cells, e.g., apopulation of amnion derived adherent cells, or a population of cellswherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells insaid isolated population of cells are amnion derived adherent cells thatsecrete one or more, or all, of VEGF, HGF, IL-8, MCP-3, FGF2,follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, uPAR, orgalectin-1, e.g., into culture medium in which the cell, or cells, aregrown.

In another embodiment, provided herein is a population of cells, e.g., apopulation of amnion derived adherent cells, or a population of cellswherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells insaid isolated population of cells are amnion derived adherent cells thatexpress angiogenic micro RNAs (miRNAs) at a higher level than bonemarrow-derived mesenchymal stem cells, wherein said miRNAs comprise oneor more, or all of, miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, and/ormiR-296. In another embodiment, provided herein is a population ofcells, e.g., a population of amnion derived adherent cells, or apopulation of cells wherein at least about 50%, 60%, 70%, 80%, 90%, 95%or 98% of cells in said isolated population of cells are amnion derivedadherent cells that express one or more of, or all of, angiogenic microRNAs (miRNAs) at a lower level than bone marrow-derived mesenchymal stemcells, wherein said miRNAs comprise one or more, or all of, miR-20a,miR-20b, miR-221, miR-222, miR-15b, and/or miR-16. In certainembodiments, AMDACs, or populations of AMDACs, express one or more, orall, the angiogenic miRNAs miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92,miR-20a, miR-20b, miR-296, miR-221, miR-222, miR-15b, and/or miR-16.

In another specific embodiment, provided herein is an amnion derivedangiogenic cell, or population of amnion derived angiogenic cells, thatexpress increased levels of CD202b, IL-8 and/or VEGF under hypoxicconditions (e.g., less than about 5% 0₂) compared to normoxic conditions(e.g., about 20% or about 21% O₂).

In another specific embodiment, the isolated population of amnionderived adherent cells additionally comprises a second type of cell. Inspecific embodiments, the AMDACs comprise at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or atleast 98% of cells in said population. In a specific embodiment, thesecond type of cell is contained within or isolated from placentalblood, umbilical cord blood, crude bone marrow or other tissues. In amore specific embodiment, said second type of cell is embryonic stemcell, a blood cell, a stem cell isolated from peripheral blood, a stemcell isolated from placental blood, a stem cell isolated from placentalperfusate, a stem cell isolated from placental tissue, a stem cellisolated from umbilical cord blood, an umbilical cord stem cell, a bonemarrow-derived mesenchymal stem cell, a mesenchymal stromal cell, ahematopoietic stem cell, a somatic stem cell, a chondrocyte, afibroblast, a muscle cell, an endothelial cell, an endothelialprogenitor cell, a pericyte, a myocyte, a cardiomyocyte, a myoblast, anangioblast, or a cardiomyoblast. In another specific embodiment, saidsecond type of cell is a hematopoietic stem or progenitor cell, e.g., aCD34⁺ cell. In another more specific embodiment, said second type ofcell comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 98% of cells in saidpopulation.

In another specific embodiment, any of the above cells are, or havebeen, proliferated in culture. In another specific embodiment, any ofthe above cells is from a culture of said cells that has been passagedat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 times, or more. In another specific embodiment, any of theabove cells is from a culture that has doubled in culture at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49 or at least 50 times, or more.

In another aspect, provided herein is a composition, e.g., apharmaceutical composition, comprising any of the amnion derivedadherent cells, or populations of cells comprising the amnion derivedadherent cells, provided herein. In a specific embodiment, thecomposition is a matrix or scaffold, e.g., a natural tissue matrix orscaffold, for example, a permanent or degradable decellularized tissuematrix or scaffold; or synthetic matrix or scaffold. In a more specificembodiment, said matrix or scaffold is shaped in the form of a tube orother three-dimensional form of an organoid. In another more specificembodiment, said matrix is a decellularized tissue matrix. In anotherspecific embodiment, the composition comprises one or more of theisolated amnion derived adherent cells provided herein, or population ofcells comprising the amnion derived adherent cells, in aphysiologically-acceptable solution, e.g., a saline solution, culturemedium or the like.

In another aspect, provided herein is a method of treating an individualhaving a disease or disorder of the circulatory system, comprisingadministering one or more of the amnion derived adherent cells describedherein to said individual in an amount and for a time sufficient fordetectable improvement of one or more symptoms of said disease ordisorder. In another embodiment, provided herein is a method of treatingan individual having a disease or disorder of the circulatory system,comprising administering amnion derived adherent cells to saidindividual in an amount and for a time sufficient for detectableimprovement of one or more indicia of cardiac function, wherein saidindicia of cardiac function are chest cardiac output (CO), cardiac index(CI), pulmonary artery wedge pressure (PAWP), cardiac index (CI), %fractional shortening (% FS), ejection fraction (EF), left ventricularejection fraction (LVEF); left ventricular end diastolic diameter(LVEDD), left ventricular end systolic diameter (LVESD), contractility(dP/dt), a decrease in atrial or ventricular functioning, an increase inpumping efficiency, a decrease in the rate of loss of pumpingefficiency, a decrease in loss of hemodynamic functioning, or decreasein complications associated with cardiomyopathy, as compared to theindividual prior to administration of amnion derived adherent cells.

In a specific embodiment, said disease or disorder is myocardialinfarction. In another specific embodiment, said disease or disorder iscardiomyopathy. In other specific embodiments, said disease or disorderis aneurysm, angina, aortic stenosis, aortitis, arrhythmias,arteriosclerosis, arteritis, asymmetric septal hypertrophy (ASH),atherosclerosis, atrial fibrillation and flutter, bacterialendocarditis, Barlow's Syndrome (mitral valve prolapse), bradycardia,Buerger's Disease (thromboangiitis obliterans), cardiomegaly, carditis,carotid artery disease, coarctation of the aorta, congenital heartdefects, congestive heart failure, coronary artery disease,Eisenmenger's Syndrome, embolism, endocarditis, erythromelalgia,fibrillation, fibromuscular dysplasia, heart block, heart murmur,hypertension, hypotension, idiopathic infantile arterial calcification,Kawasaki Disease (mucocutaneous lymph node syndrome, mucocutaneous lymphnode disease, infantile polyarteritis), metabolic syndrome,microvascular angina, myocarditis, paroxysmal atrial tachycardia (PAT),periarteritis nodosa (polyarteritis, polyarteritis nodosa),pericarditis, peripheral vascular disease, critical limb ischemia,phlebitis, pulmonary valve stenosis (pulmonic stenosis), Raynaud'sDisease, renal artery stenosis, renovascular hypertension, rheumaticheart disease, diabetic vasculopathy, septal defects, silent ischemia,syndrome X, tachycardia, Takayasu's Arteritis, Tetralogy of Fallot,transposition of the great vessels, tricuspid atresia, truncusarteriosus, valvular heart disease, varicose ulcers, varicose veins,vasculitis, ventricular septal defect, Wolff-Parkinson-White Syndrome,endocardial cushion defect, acute rheumatic fever, acute rheumaticpericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis,chronic rheumatic heart diseases, diseases of the mitral valve, mitralstenosis, rheumatic mitral insufficiency, diseases of aortic valve,diseases of other endocardial structures, ischemic heart disease (acuteand subacute), angina pectoris, acute pulmonary heart disease, pulmonaryembolism, chronic pulmonary heart disease, kyphoscoliotic heart disease,myocarditis, endocarditis, endomyocardial fibrosis, endocardialfibroelastosis, atrioventricular block, cardiac dysrhythmias, myocardialdegeneration, cerebrovascular disease, a disease of arteries, arteriolesand capillaries, or a disease of veins and lymphatic vessels.

In other specific embodiments, said disease or disorder is an occlusionand stenosis of precerebral arteries, or occlusion of cerebral arteries.In one aspect, provided herein is a method of treating an individual whohas a disruption of the flow of blood in or around the individual'sbrain, e.g., who has a symptom or neurological deficit attributable to adisruption of the flow of blood in or around the individual's brain orcentral nervous system (CNS), comprising administering to saidindividual a therapeutically effective amount of isolated AMDACs. Incertain embodiments, the disruption of flow of blood results in anoxicinjury or hypoxic injury to the individual's brain or CNS.

In other specific embodiments, said disease or disorder is an occlusionand stenosis of peripheral arteries. In one aspect, provided herein is amethod of treating an individual who has a disruption of the flow ofblood in or around limb, e.g., who has a symptom or vascular deficitattributable to a disruption of the flow of blood in or around theindividual's peripheral vascular system, comprising administering tosaid individual a therapeutically effective amount of isolated AMDACs.In certain embodiments, the disruption of flow of blood results inanoxic injury or hypoxic injury to the individual's limbs and orextremities.

In another aspect, provided herein is a method of treating an individualsuffering from a wound or trauma, comprising administering one or moreof the amnion derived adherent cells described herein to said individualin an amount and for a time sufficient for detectable improvement ofsaid wound or trauma.

In another specific embodiment of the method of treatment, said cellsare administered to said individual by injection. In a more specificembodiment, said injection is injection into an ischemic area of theindividual's heart. In another specific embodiment of the method oftreatment, said cells are administered to said individual by intravenousinfusion. In another specific embodiment of the method of treatment,said cells, or a population of said cells, or a population of cellscomprising said cells, is administered to said individual byimplantation in said individual of a matrix or scaffold comprisingamnion derived adherent cells, as described above.

The isolated amnion derived adherent cells and cell populations providedherein are not the isolated placental stem cells or cell populationsdescribed, e.g., in U.S. Pat. No. 7,255,879 or U.S. Patent ApplicationPublication No. 2007/0275362. The isolated amnion derived adherent cellsprovided herein are also not endothelial progenitor cells, amnioticepithelial cells, trophoblasts, cytotrophoblasts, embryonic germ cells,embryonic stem cells, cells obtained from the inner cell mass of anembryo, or cells obtained from the gonadal ridge of an embryo.

As used herein, the term “about” means, e.g., within 10% of a statedfigure or value.

As used herein, the term “angiogenic,” in reference to the amnionderived adherent cells described herein, means that the cells can formvessels or vessel-like sprouts, or that the cells can promoteangiogenesis (e.g., the formation of vessels or vessel-like structures)in another population of cells, e.g., endothelial cells.

As used herein, the term “angiogenesis” refers to the process of bloodvessel formation that includes, but is not limited to, endothelial cellactivation, migration, proliferation, matrix remodeling and cellstabilization.

As used herein, the term “stem cell” defines the functional propertiesof any given cell population that can proliferate extensively, but notnecessarily infinitely, and contribute to the formation of multipletissues, either during embryological development or post-natal tissuereplacement and repair.

As used herein, the term “progenitor cell” defines the functionalproperties of any given cell population that can proliferateextensively, but not necessarily infinitely, and contribute to theformation of a restricted set of multiple tissues in comparison to astem cell, either during embryological development or post-natal tissuereplacement and repair.

As used herein, the term “derived” means isolated from or otherwisepurified. For example, amnion derived adherent cells are isolated fromamnion. The term “derived” encompasses cells that are cultured fromcells isolated directly from a tissue, e.g., the amnion, and cellscultured or expanded from primary isolates.

As used herein, “immunolocalization” means the detection of a compound,e.g., a cellular marker, using an immune protein, e.g., an antibody orfragment thereof in, for example, flow cytometry, fluorescence-activatedcell sorting, magnetic cell sorting, in situ hybridization,immunohistochemistry, or the like.

As used herein, the term “isolated cell” means a cell that issubstantially separated from other, cells of the tissue, e.g., amnion orplacenta, from which the cell is derived. A cell is “isolated” if atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99%of the cells with which the stem cell is naturally associated areremoved from the cell, e.g., during collection and/or culture of thecell.

As used herein, the term “isolated population of cells” means apopulation of cells that is substantially separated from other cells ofthe tissue, e.g., amnion or placenta, from which the population of cellsis derived. A cell is “isolated” if at least about 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with which thepopulation of cells, or cells from which the population of cells isderived, is naturally associated are removed from the cell, e.g., duringcollection and/or culture of amnion derived adherent cells.

As used herein, a cell is “positive” for a particular marker when thatmarker is detectable above background, e.g., by immunolocalization,e.g., by flow cytometry; or by RT-PCR. For example, a cell is describedas positive for, e.g., CD105 if CD105 is detectable on the cell in anamount detectably greater than background (in comparison to, e.g., anisotype control). In the context of, e.g., antibody-mediated detection,“positive,” as an indication a particular cell surface marker ispresent, means that the marker is detectable using an antibody, e.g., afluorescently-labeled antibody, specific for that marker; “positive”also means that a cell bears that marker in a amount that produces asignal, e.g., in a cytometer, that is detectably above background. Forexample, a cell is “CD105⁺” where the cell is detectably labeled with anantibody specific to CD105, and the signal from the antibody isdetectably higher than a control (e.g., background). Conversely,“negative” in the same context means that the cell surface marker is notdetectable using an antibody specific for that marker compared tobackground. For example, a cell is “CD34⁻” where the cell is notdetectably labeled with an antibody specific to CD34. Unless otherwisenoted herein, cluster of differentiation (“CD”) markers are detectedusing antibodies. For example, OCT-4 can be determined to be present,and a cell is OCT-4⁺, if mRNA for OCT-4 is detectable using RT-PCR,e.g., for 30 cycles.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows expression of stem cell-related genes by amnion derivedadherent cells and NTERA-2 cells.

FIGS. 2A-2B show the expression of TEM-7 on the cell surface of amnionderived adherent cells (AMDACs). 2A: Staining of AMDACs with anti-TEM-7;2B: staining of AMDACs with isotype control.

FIGS. 3A-3D show the secretion of selected angiogenic proteins by amnionderived adherent cells. 3A: secretion of TIMP-1, thrombopoietin, VEGF,VEGF-D by amnion derived adherent cells; 3B: secretion of angiogenin,EGF, ENA-78, bFGF, GRO by amnion derived adherent cells; 3C: secretionof IFN-gamma, IGF-I, IL-6, IL-8, leptin by amnion derived adherentcells; 3D: secretion of MCP-1, PDGF-BB, PIGF, RANTES, TGF-beta 1 byamnion derived adherent cells.

FIG. 4 shows the angiogenic effect of amnion derived adherent cellsconditioned medium on Human Endothelial Cell (HUVEC) tube formation.

FIG. 5 shows the angiogenic effect of amnion derived adherent cellsconditioned medium on human endothelial cell migration.

FIG. 6 shows the effect of amnion derived adherent cell-conditionedmedium on Human Endothelial Cell proliferation.

FIGS. 7A-7C show the uptake of acetylated LDL by HUVECs (7A) and amnionderived adherent cells without VEGF (7B) and with VEGF (7C).

FIGS. 8A-8C show tube formation of HUVECs (8A) and amnion derivedadherent cells without VEGF (8B) and with VEGF (8C).

FIGS. 9A-9B show the secretion of VEGF (9A) and IL-8 (9B) by amnionderived adherent cells under hypoxic and normoxic conditions.

FIG. 10 shows the expression of cellular marker Tie2 under normoxic(about 21% O₂) and hypoxic (less than about 5% O₂) conditions. Y axis:percentage of cells positive for Tie2 by flow cytometry.

FIG. 11 shows the cardiomyocytic differentiation potency of amnionderived adherent cells, wherein AM refers to amnion derived adherentcells (AMDACs), HD refers to untreated hanging drop, HD IND refers tohanging drop exposed to inducing conditions, and HD IND+5-AZA refers toinduction in the presence or absence of 5-azacytidine. CTRL refers tothe untreated hanging drop control.

FIG. 12 shows positive effect of AMDACs on angiogenesis in a chickchorioallantois angiogenesis model. Lot 1, Lot 2, Lot 3: AMDACs fromthree separate cell preparations. bFGF: basic fibroblast growth factor(positive control). MDAMB231: Angiogenic breast cancer cell line(positive control). Y axis: Degree of blood vessel formation.

FIG. 13 shows positive effect of AMDAC-conditioned medium onangiogenesis in a chick chorioallantois angiogenesis model. Lot 1, Lot2, Lot 3: AMDACs from three separate cell preparations. bFGF: basicfibroblast growth factor (positive control). MDAMB231: Angiogenic breastcancer cell line (positive control). Y axis: Degree of blood vesselformation.

FIGS. 14A, 14B: Hydrogen peroxide-generated reactive oxygen speciespresent in cultures of astrocytes, co-cultures of astrocytes and bonemarrow-derived mesenchymal stem cells (BM-MSCs), or co-cultures ofastrocytes and AMDACs. 14A: AMDACs, Lot 1; 14B: AMDACs, Lot 2. Theconditions HA (human astrocytes) alone, astrocytes+H₂O₂, andastrocytes+BM-MSCs+H₂O₂ are the same for FIGS. 14A and 14B. RFU ROSactivity: Relative fluorescence units for reactive oxygen species.

5. DETAILED DESCRIPTION

5.1 Characteristics of Amnion Derived Adherent Cells

Provided herein are unique adherent, angiogenic cells, and populationsof such cells, isolatable from the amnion, referred to herein as “amnionderived adherent cells” or AMDACs. Amnion derived adherent cellssuperficially resemble mesenchymal cells in appearance, having agenerally fibroblastoid shape. The cells adhere to a cell culturesurface, e.g., to tissue culture plastic.

AMDACs display cellular markers that distinguish them from otheramnion-derived, or placenta-derived, cells. For example, in oneembodiment, the amnion derived adherent cell is OCT-4⁻ (octamer bindingprotein 4), as determined by RT-PCR. In another specific embodiment, theOCT-4⁻ amnion derived adherent cell is CD49f⁺, as determined byimmunolocalization. In another specific embodiment, said OCT-4⁻ cell isHLA-G⁻, as determined by RT-PCR. In another specific embodiment, theOCT-4⁻ cell is VEGFR1/Flt-1⁺ (vascular endothelial growth factorreceptor 1) and/or VEGFR2/KDR⁺ (vascular endothelial growth factorreceptor 2), as determined by immunolocalization. In a specificembodiment, the OCT-4⁻ amnion derived adherent cell, or a population ofOCT-4⁻ amnion derived adherent cells, expresses at least 2 log lessPCR-amplified mRNA for OCT-4 at, e.g., 20 cycles, than an NTERA-2 cell,or population of NTERA-2 cells having an equivalent number of cells andRNA amplification cycles. In another specific embodiment, said OCT-4⁻cell is CD90⁺, CD105⁺, or CD117⁻. In a more specific embodiment, saidOCT-4⁻ cell is CD90⁺, CD105⁺, and CD117⁻. In a more specific embodiment,the cell is OCT-4⁻ or HLA-G⁻, and is additionally CD49f⁻, CD90⁺, CD105⁺,and CD117⁻. In a more specific embodiment, the cell is OCT-4⁻, HLA-G⁻,CD49f⁺, CD90⁺, CD105⁺, and CD117⁻. In another specific embodiment, theOCT-4⁻ cell does not express SOX2, e.g., as determined by RT-PCR for 30cycles. In a specific embodiment, therefore, the cell is OCT-4⁻, CD49f⁺,CD90⁺, CD105⁺, and CD117⁻, as determined by immunolocalization or flowcytometry, and SOX2⁻, as determined by RT-PCR, e.g., for 30 cycles.

In another embodiment, said OCT-4⁻ cell is one or more of CD29⁺, CD73⁺,ABC-p⁺, and CD38⁻, as determined by immunolocalization.

In another specific embodiment, for example, an OCT-4⁻ AMDAC canadditionally be one or more of CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, TEM-7⁺(tumor endothelial marker 7), CD31⁻, CD34⁻, CD45⁻, CD133⁻, CD143⁻(angiotensin-I-converting enzyme, ACE), CD146⁻ (melanoma cell adhesionmolecule), or CXCR4⁻ (chemokine (C-X-C motif) receptor 4) as determinedby immunolocalization, or HLA-G⁻ as determined by RT-PCR. In a morespecific embodiment, said cell is CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺,Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻, and CXCR4⁻as determined by immunolocalization, and HLA-G⁻ as determined by RT-PCR.In one embodiment, the amnion derived adherent cell provided herein isone or more of CD31⁻, CD34⁻, CD45⁻, and/or CD133⁻. In a specificembodiment, the amnion derived adherent cell is OCT-4⁻, as determined byRT-PCR; VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺, as determined byimmunolocalization; and one or more, or all, of CD31⁻, CD34⁻, CD45⁻,and/or CD133⁻.

In another specific embodiment, said cell is additionally VE-cadherin⁻as determined by immunolocalization. In another specific embodiment,said cell is additionally positive for CD105⁺ and CD200⁺ as determinedby immunolocalization. In another specific embodiment, said cell doesnot express CD34 as detected by immunolocalization after exposure to 1to 100 ng/mL VEGF for 4 to 21 days. In more specific embodiments, saidcell does not express CD34 as detected by immunolocalization afterexposure to 25 to 75 ng/mL VEGF for 4 to 21 days, or to 50 ng/mL VEGFfor 4 to 21 days. In even more specific embodiments, said cell does notexpress CD34 as detected by immunolocalization after exposure to 1, 2.5,5, 10, 25, 50, 75 or 100 ng/mL VEGF for 4 to 21 days. In yet morespecific embodiments, said cell does not express CD34 as detected byimmunolocalization after exposure to 1 to 100 ng/mL VEGF for 7 to 14,e.g., 7, days.

In specific embodiments, the amnion derived adherent cell is OCT-4⁻, asdetermined by RT-PCR, and one or more of VE-cadherin⁻, VEGFR2/KDR⁺,CD9⁺, CD54⁺, CD105⁺, and/or CD200⁺ as determined by immunolocalization.In a specific embodiment, the amnion derived cell is OCT-4⁻, asdetermined by RT-PCR, and VE-cadherin⁻, VEGFR2/KDR⁺, CD9⁺, CD54⁺,CD105⁺, and CD200⁺ as determined by immunolocalization. In anotherspecific embodiment, said cells do not express CD34, as detected byimmunolocalization, e.g., after exposure to 1 to 100 ng/mL VEGF for 4 to21 days.

In another embodiment, the amnion derived adherent cell is OCT-4⁻,CD49f⁻, HLA-G⁻, CD90⁺, CD105⁺, and CD117⁻. In a more specificembodiment, said cell is one or more of CD9⁺, CD10⁺, CD44⁺, CD54⁺,CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻, orCXCR4⁻, as determined by immunolocalization. In a more specificembodiment, said cell is CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, Tie-2⁺,TEM-7⁺, CD31⁻, CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻, and CXCR4⁻ asdetermined by immunolocalization. In another specific embodiment, saidcell is additionally VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺, as determined byimmunolocalization; and one or more of CD31⁻, CD34⁻, CD45⁻, CD133⁻,and/or Tie-2⁻ as determined by immunolocalization. In another specificembodiment, said cell is additionally VEGFR1/Flt-1⁺, VEGFR2/KDR⁺, CD31⁻,CD34⁻, CD45⁻, CD133⁻, and Tie-2⁻ as determined by immunolocalization.

In another embodiment, the OCT-4− amnion derived adherent cells areadditionally one or more, or all, of CD9⁺, CD10⁺, CD44⁺, CD49f⁺, CD54⁺,CD90⁺, CD98⁺, CD105⁺, CD200⁺, Tie-2⁺, TEM-7⁺, VEGFR1/Flt-1⁺, and/orVEGFR2/KDR⁺ (CD309⁺), as determined by immunolocalization; oradditionally one or more, or all, of CD31⁻, CD34⁻, CD38⁻, CD45⁻, CD117⁻,CD133⁻, CD143⁻, CD144⁻, CD146⁻, CD271⁻, CXCR4⁻, HLA-G⁻, and/orVE-cadherin⁻, as determined by immunolocalization, or SOX2⁻, asdetermined by RT-PCR.

In certain embodiments, the isolated tissue culture plastic-adherentamnion derived adherent cells are CD49f⁺. In a specific embodiment, saidCD49f⁺ cells are additionally one or more, or all, of CD9⁺, CD10⁺,CD44⁺, CD54⁺, CD90⁺, CD98⁺, CD105⁺, CD200⁺, Tie-2⁺, TEM-7⁺,VEGFR1/Flt-1⁺, and/or VEGFR2/KDR⁺ (CD309⁺), as determined byimmunolocalization; or additionally one or more, or all, of CD31⁻,CD34⁻, CD38⁻, CD45⁻, CD117⁻, CD133⁻, CD143⁻, CD144⁻, CD146⁻, CD271⁻,CXCR4⁻, HLA-G⁻, OCT-4⁻ and/or VE-cadherin⁻, as determined byimmunolocalization, or SOX2⁻, as determined by RT-PCR.

In certain other embodiments, the isolated tissue cultureplastic-adherent amnion derived adherent cells are HLA-G⁻, CD90⁺, andCD117⁻. In a specific embodiment, said HLA-G⁻, CD90⁺, and CD117⁻ cellsare additionally one or more, or all, of CD9⁺, CD10⁺, CD44⁺, CD49f⁻,CD54⁺, CD98⁺, CD105⁺, CD200⁺, Tie-2⁺, TEM-7⁺, VEGFR1/Flt-1⁺, and/orVEGFR2/KDR⁺ (CD309⁺), as determined by immunolocalization; oradditionally one or more, or all, of CD31⁻, CD34⁻, CD38⁻, CD45⁻, CD133⁻,CD143⁻, CD144⁻, CD146⁻, CD271⁻, CXCR4⁻, OCT-4⁻ and/or VE-cadherin⁻, asdetermined by immunolocalization, or SOX2⁻, as determined by RT-PCR.

In another embodiment, the isolated amnion derived adherent cells, orpopulation of amnion derived angiogenic cells, do not constitutivelyexpress mRNA for fibroblast growth factor 4 (FGF4), interferon γ (IFNG),chemokine (C-X-C motif) ligand 10 (CXCL10), angiopoietin 4 (ANGPT4),angiopoietin-like 3 (ANGPTL3), fibrinogen a chain (FGA), leptin (LEP),prolactin (PRL), prokineticin 1 (PROK1), tenomodulin (TNMD), FMS-liketyrosine kinase 3 (FLT3), extracellular link domain containing 1(XLKD1), cadherin 5, type 2 (CDH5), leukocyte cell derived chemotaxin 1(LECT1), plasminogen (PLG), telomerase reverse transcriptase (TERT),(sex determining region Y)-box 2 (SOX2), NANOG, matrix metalloprotease13 (MMP-13), distal-less homeobox 5 (DLX5), and/or bonegamma-carboxyglutamate (gla) protein (BGLAP), as determined by RT-PCR,e.g., for 30 cycles under standard culture conditions. In otherembodiments, isolated amnion derived adherent cells, or population ofamnion derived angiogenic cells, express mRNA for (ARNT2), nerve growthfactor (NGF), brain-derived neurotrophic factor (BDNF), glial-derivedneurotrophic factor (GDNF), neurotrophin 3 (NT-3), NT-5,hypoxia-Inducible Factor 1α (HIF1A), hypoxia-inducible protein 2 (HIG2),heme oxygenase (decycling) 1 (HMOX1), Extracellular superoxide dismutase[Cu—Zn] (SOD3), catalase (CAT), transforming growth factor β1 (TGFB1),transforming growth factor β1 receptor (TGFB1R), and hepatoycte growthfactor receptor (HGFR/c-met)

In another aspect, provided herein are isolated populations of cellscomprising the amnion derived adherent cells described herein. Thepopulations of cells can be homogeneous populations, e.g., a populationof cells, at least about 90%, 95%, 98% or 99% of which are amnionderived adherent cells. The populations of cells can be heterogeneous,e.g., a population of cells wherein at most about 10%, 20%, 30%, 40%,50%, 60%, 70% or 80% of the cells in the population are amnion derivedadherent cells. The isolated populations of cells are not, however,tissue, i.e., amniotic membrane.

In one embodiment, provided herein is an isolated population of cellscomprising AMDACs, e.g., a population of cells substantially homogeneousfor AMDACs, wherein said AMDACs are adherent to tissue culture plastic,and wherein said AMDACs are OCT-4⁻, as determined by RT-PCR. In aspecific embodiment, the AMDACs are CD49f⁺ or HLA-G⁺, e.g., asdetermined by immunolocalization or RT-PCR. In another specificembodiment, said population of AMDACs is VEGFR1/Flt-1⁺ and/orVEGFR2/KDR⁺ as determined by immunolocalization, wherein said isolatedpopulation of cells is not an amnion or amniotic membrane. In a morespecific embodiment, the AMDACs are OCT-4⁻, and/or HLA-G⁻ as determinedby RT-PCR, and VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺ as determined byimmunolocalization. In a specific embodiment, at least about 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% of cells in said population are saidamnion derived adherent cells. In another specific embodiment, saidAMDACs are CD90⁺, CD105⁺, or CD117⁻. In a more specific embodiment, saidAMDACs are CD90⁺, CD105⁺, and CD117⁻. In a more specific embodiment, theAMDACs are OCT-4⁻, CD49f⁻, CD90⁺, CD105⁺, and CD117⁻. In anotherspecific embodiment, the AMDACs do not express SOX2, e.g., as determinedby RT-PCR for 30 cycles. In an even more specific embodiment, thepopulation comprises AMDACs, wherein said AMDACs are OCT-4⁻, HLA-G⁻,CD49f⁺, CD90⁺, CD105⁺, and CD117⁻, as determined by immunolocalizationor flow cytometry, and SOX2⁻, e.g., as determined by RT-PCR for 30cycles

In another specific embodiment, said AMDACs in said population of cellsare CD90⁺, CD105⁺, or CD117⁻, as determined by immunolocalization orflow cytometry. In a more specific embodiment, the AMDACs are CD90⁺,CD105⁺, and CD117⁻, as determined by immunolocalization or flowcytometry. In a more specific embodiment, the AMDACs are OCT-4⁻ orHLA-G⁻, e.g., as determined by RT-PCR, and are additionally CD49f⁺,CD90⁺, CD105⁺, and CD117⁻ as determined by immunolocalization or flowcytometry. In a more specific embodiment, the AMDACs in said populationof cells are OCT-4⁻, HLA-G⁻, CD49f⁻, CD90⁺, CD105⁺, and CD117⁻. Inanother specific embodiment, the AMDACs do not express SOX2, e.g., asdetermined by RT-PCR for 30 cycles. In a more specific embodiment,therefore, the cell is OCT-4⁻, CD49f⁺, CD90⁺, CD105⁺, and CD117⁻, asdetermined by immunolocalization or flow cytometry, and SOX2⁻, asdetermined by RT-PCR, e.g., for 30 cycles. In an even more specificembodiment, the AMDACs are OCT-4⁻ or HLA-G⁻, and are additionallyCD49f⁻, CD90⁺, CD105⁺, and CD117⁻. In a more specific embodiment, theAMDACs are OCT-4⁻, HLA-G⁻, CD49f⁺, CD90⁺, CD105⁺, and CD117⁻.

In another embodiment, the amnion derived adherent cells in saidpopulation of cells are adherent to tissue culture plastic, OCT-4⁻ asdetermined by RT-PCR, and VEGFR1/Flt-1⁺ and/or VEGFR2/KDR⁺ as determinedby immunolocalization, and are additionally one or more of CD9⁺, CD10⁺,CD44⁺, CD54⁺, CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻, CD45⁻, CD133⁻,CD143⁻, CD146⁻, or CXCR4⁻, as determined by immunolocalization, orHLA-G⁻ as determined by RT-PCR, and wherein said isolated population ofcells is not an amnion. In another embodiment, provided herein is anisolated population of cells comprising an amnion derived adherent cell,wherein said cell is adherent to tissue culture plastic, wherein saidcell is OCT-4⁻ as determined by RT-PCR, and VEGFR1/Flt-1⁺ and/orVEGFR2/KDR⁺ as determined by immunolocalization, wherein said cell doesnot express CD34 as detected by immunolocalization after exposure to 1to 100 ng/mL VEGF for 4 to 21 days, and wherein said isolated populationof cells is not an amnion. In a specific embodiment of any of the aboveembodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% ofcells in said population are said amnion derived adherent cells.

In another embodiment, any of the above populations of cells comprisingamnion derived adherent cells forms sprouts or tube-like structures whencultured in the presence of an extracellular matrix protein, e.g., likecollagen type I and IV, or an angiogenic factor, e.g., like vascularendothelial growth factor (VEGF), epithelial growth factor (EGF),platelet derived growth factor (PDGF) or basic fibroblast growth factor(bFGF), e.g., in or on a substrate such as placental collagen, e.g., orMATRIGELTm for at least 4 days and up to 14 days.

Amnion derived adherent cells, and populations of amnion derivedadherent cells, display characteristic expression of proteins related toangiogenesis-related or cardiomyogenesis-related genes. In certainembodiments, provided herein is a cell that expresses, or a populationof cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% ofcells in said isolated population of cells are amnion derived adherentcells that express RNA for one or more of, or all of, ACTA2 (actin,alpha 2, smooth muscle, aorta), ADAMTS1 (ADAM metallopeptidase withthrombospondin type 1 motif, 1), AMOT (angiomotin), ANG (angiogenin),ANGPT1 (angiopoietin 1), ANGPT2, ANGPTL1 (angiopoietin-like 1), ANGPTL2,ANGPTL4, BAI1 (brain-specific angiogenesis inhibitor 1), CD44, CD200,CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1),CHGA (chromogranin A), COL15A1 (collagen, type XV, alpha 1), COL18A1(collagen, type XVIII, alpha 1), COL4A1 (collagen, type IV, alpha 1),COL4A2 (collagen, type IV, alpha 2), COL4A3 (collagen, type IV, alpha3), CSF3 (colony stimulating factor 3 (granulocyte), CTGF (connectivetissue growth factor), CXCL12 (chemokine (CXC motif) ligand 12 (stromalcell-derived factor 1)), CXCL2, DNMT3B (DNA(cytosine-5-)-methyltransferase 3 beta), ECGF1 (thymidinephosphorylase), EDG1 (endothelial cell differentiation gene 1), EDIL3(EGF-like repeats and discoidin I-like domains 3), ENPP2 (ectonucleotidepyrophosphatase/phosphodiesterase 2), EPHB2 (EPH receptor B2), FBLN5(FIBULIN 5), F2 (coagulation factor II (thrombin)), FGF1 (acidicfibroblast growth factor), FGF2 (basic fibroblast growth factor), FIGF(c-fos induced growth factor (vascular endothelial growth factor D)),FLT4 (fms-related tyrosine kinase 4), FN1 (fibronectin 1), FST(follistatin), FOXC2 (forkhead box C2 (MFH-1, mesenchyme forkhead 1)),GRN (granulin), HGF (hepatocyte growth factor), HEY1(hairy/enhancer-of-split related with YRPW motif 1), HSPG2 (heparansulfate proteoglycan 2), IFNB1 (interferon, beta 1, fibroblast), IL8(interleukin 8), IL12A, ITGA4 (integrin, alpha 4; CD49d), ITGAV(integrin, alpha V), ITGB3 (integrin, beta 3), MDK (midkine), MMP2(matrix metalloprotease 2), MYOZ2 (myozenin 2), NRP1 (neuropilin 1),NRP2, PDGFB (platelet-derived growth factor (3), PDGFRA(platelet-derived growth factor receptor a), PDGFRB, PECAM1(platelet/endothelial cell adhesion molecule), PF4 (platelet factor 4),PGK1 (phosphoglycerate kinase 1), PROX1 (prospero homeobox 1), PTN(pleiotrophin), SEMA3F (semophorin 3F), SERPINB5 (serpin peptidaseinhibitor, clade B (ovalbumin), member 5), SERPINC1, SERPINF1, TIMP2(tissue inhibitor of metalloproteinases 2), TIMP3, TGFA (transforminggrowth factor, alpha), TGFB1, THBS1 (thrombospondin 1), THBS2, TIE1(tyrosine kinase with immunoglobulin-like and EGF-like domains 1),TIE2/TEK, TNF (tumor necrosis factor), TNNI1 (troponin I, type 1),TNFSF15 (tumor necrosis factor (ligand) superfamily, member 15), VASH1(vasohibin 1), VEGF (vascular endothelial growth factor), VEGFB, VEGFC,VEGFR1/FLT1 (vascular endothelial growth factor receptor 1), and/orVEGFR2/KDR.

When human cells are used, the gene designations throughout refer tohuman sequences, and, as is well known to persons of skill in the art,representative sequences can be found in literature, or in GenBank.Probes to the sequences can be determined by sequences that arepublicly-available, or through commercial sources, e.g., specificTAQMAN® probes or TAQMAN® Angiogenesis Array (Applied Biosystems, partno. 4378710).

Amnion derived adherent cells, and populations of amnion derivedadherent cells, display characteristic expression ofangiogenesis-related proteins. In certain embodiments, provided hereinis a cell that expresses, or a population of cells, wherein at leastabout 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells in said isolatedpopulation of cells are amnion derived adherent cells that expressCD49d, Connexin-43, HLA-ABC, Beta 2-microglobulin, CD349, CD318, PDL1,CD106, Galectin-1, ADAM 17 precursor (A disintegrin andmetalloproteinase domain 17) (TNF-alpha converting enzyme) (TNF-alphaconvertase), Angiotensinogen precursor, Filamin A (Alpha-filamin)(Filamin 1) (Endothelial actin-binding protein) (ABP-280) (Nonmusclefilamin), Alpha-actinin 1 (Alpha-actinin cytoskeletal isoform)(Non-muscle alpha-actinin 1) (F-actin cross linking protein),Low-density lipoprotein receptor-related protein 2 precursor (Megalin)(Glycoprotein 330) (gp330), Macrophage scavenger receptor types I and II(Macrophage acetylated LDL receptor I and II), Activin receptor type IIBprecursor (ACTR-IIB), Wnt-9 protein, Glial fibrillary acidic protein,astrocyte (GFAP), Myosin-binding protein C, cardiac-type (CardiacMyBP-C) (C-protein, cardiac muscle isoform), and/or Myosin heavy chain,nonmuscle type A (Cellular myosin heavy chain, type A) (Nonmuscle myosinheavy chain-A) (NMMHC-A).

The amnion derived adherent cells provided herein further secreteproteins that promote angiogenesis, e.g., in endothelial cells,endothelial progenitor cells, or the like. In certain embodiments, theamnion derived adherent cell, population of amnion derived adherentcells, or population of cells comprising amnion derived adherent cells,e.g., wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% ofcells in said isolated population of cells are amnion derived adherentcells, secrete one or more, or all, of VEGF, HGF, IL-8, MCP-3, FGF2,Follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2,uPAR, Galectin-1, e.g., into culture medium in which the cell, or cells,are grown.

In another embodiment, any of the above populations of cells comprisingamnion derived adherent cells can cause the formation of sprouts ortube-like structures in a population of endothelial cells in contactwith said amnion derived adherent cells. In a specific embodiment, theamnion-derived angiogenic cells are co-cultured with human endothelialcells, forming sprouts or tube-like structures, or supporting theendothelial cell sprouts, e.g., when cultured in the presence ofextracellular matrix proteins such as collagen type I and IV, and/orangiogenic factors such as vascular endothelial growth factor (VEGF),epithelial growth factor (EGF), platelet derived growth factor (PDGF) orbasic fibroblast growth factor (bFGF), e.g., in or on a substrate suchas placental collagen or MATRIGEL™ for at least 4 days and/or up to 14days.

In another embodiment, any of the above populations of cells comprisingamnion derived adherent cells secrete angiogenic factors such asvascular endothelial growth factor (VEGF), epithelial growth factor(EGF), platelet derived growth factor (PDGF), basic fibroblast growthfactor (bFGF), or Interleukin-8 (IL-8) and thereby can induce humanendothelial cells to form sprouts or tube-like structures when culturedin the presence of extracellular matrix proteins such as collagen type Iand IV e.g., in or on a substrate such as placental collagen orMATRIGELTm.

In another embodiment, provided herein is a population of cells, e.g., apopulation of amnion derived adherent cells, or a population of cellswherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells insaid isolated population of cells are amnion derived adherent cells thatexpress angiogenic micro RNAs (miRNAs) at a higher level than bonemarrow-derived mesenchymal stem cells, wherein said miRNAs comprise oneor more, or all of, miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, and/ormiR-296. In another embodiment, provided herein is a population ofcells, e.g., a population of amnion derived adherent cells, or apopulation of cells wherein at least about 50%, 60%, 70%, 80%, 90%, 95%or 98% of cells in said isolated population of cells are amnion derivedadherent cells that express one or more of, or all of, angiogenic microRNAs (miRNAs) at a lower level than bone marrow-derived mesenchymal stemcells, wherein said miRNAs comprise one or more, or all of, miR-20a,miR-20b, miR-221, miR-222, miR-15b, and/or miR-16. In certainembodiments, AMDACs, or populations of AMDACs, express one or more, orall, of the angiogenic miRNAs miR-17-3p, miR-18a, miR-18b, miR-19b,miR-92, miR-20a, miR-20b, (members of the of the angiogenic miRNAcluster 17-92), miR-296, miR-221, miR-222, miR-15b, and/or miR-16.

Thus, in one embodiment, provided herein is an isolated amnion derivedadherent cell, wherein said cell is adherent to tissue culture plastic,and wherein said cell is OCT-4⁻, as determined by RT-PCR, and CD49f⁺,HLA-G⁻, CD90⁺, CD105⁺, and CD117⁻, as determined by immunolocalization,and wherein said cell: (a) expresses one or more of CD9, CD10, CD44,CD54, CD98, CD200, Tie-2, TEM-7, VEGFR1/Flt-1, or VEGFR2/KDR (CD309), asdetermined by immunolocalization; (b) lacks expression of CD31, CD34,CD38, CD45, CD133, CD143, CD144, CD146, CD271, CXCR4, HLA-G, orVE-cadherin, as determined by immunolocalization, or lacks expression ofSOX2, as determined by RT-PCR; (c) express mRNA for ACTA2, ADAMTS1,AMOT, ANG, ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL4, BAIL CD44, CD200,CEACAM1, CHGA, COL15A1, COL18A1, COL4A1, COL4A2, COL4A3, CSF3, CTGF,CXCL12, CXCL2, DNMT3B, ECGF1, EDG1, EDIL3, ENPP2, EPHB2, FBLN5, F2,FGF1, FGF2, FIGF, FLT4, FN1, FST, FOXC2, GRN, HGF, HEY1, HSPG2, IFNB1,IL8, IL12A, ITGA4, ITGAV, ITGB3, MDK, MMP2, MYOZ2, NRP1, NRP2, PDGFB,PDGFRA, PDGFRB, PECAM1, PF4, PGK1, PROX1, PTN, SEMA3F, SERPINB5,SERPINC1, SERPINF1, TIMP2, TIMP3, TGFA, TGFB1, THBS1, THBS2, TIE1,TIE2/TEK, TNF, TNNI1, TNFSF15, VASH1, VEGF, VEGFB, VEGFC, VEGFR1/FLT1,or VEGFR2/KDR; (d) expresses one or more of the proteins CD49d,Connexin-43, HLA-ABC, Beta 2-microglobulin, CD349, CD318, PDL1, CD106,Galectin-1, ADAM 17, angiotensinogen precursor, filamin A, alpha-actinin1, megalin, macrophage acetylated LDL receptor I and II, activinreceptor type IIB precursor, Wnt-9 protein, glial fibrillary acidicprotein, astrocyte, myosin-binding protein C, or myosin heavy chain,nonmuscle type A; (e) secretes VEGF, HGF, IL-8, MCP-3, FGF2,Follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2,uPAR, or galectin-1 into culture medium in which the cell grows; (f)expresses micro RNAs miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, ormiR-296 at a higher level than an equivalent number of bonemarrow-derived mesenchymal stem cells; (g) expresses micro RNAs miR-20a,miR-20b, miR-221, miR-222, miR-15b, or miR-16 at a lower level than anequivalent number of bone marrow-derived mesenchymal stem cells; (h)expresses miRNAs miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a,miR-20b, miR-296, miR-221, miR-222, miR-15b, or miR-16; and/or (i)expresses increased levels of CD202b, IL-8 or VEGF when cultured in lessthan about 5% O₂, compared to expression of CD202b, IL-8 or VEGF under21% O₂. In a specific embodiment, the isolated amnion derived adherentcell is OCT-4⁻, as determined by RT-PCR, and CD49f⁺, HLA-G⁻, CD90⁺,CD105⁺, and CD117⁻, as determined by immunolocalization, and (a)expresses CD9, CD10, CD44, CD54, CD90, CD98, CD200, Tie-2, TEM-7,VEGFR1/Flt-1, and/or VEGFR2/KDR (CD309), as determined byimmunolocalization; (b) lacks expression of CD31, CD34, CD38, CD45,CD133, CD143, CD144, CD146, CD271, CXCR4, HLA-G, and/or VE-cadherin, asdetermined by immunolocalization, or lacks expression of SOX2, asdetermined by RT-PCR; (c) express mRNA for ACTA2, ADAMTS1, AMOT, ANG,ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL4, BAI1, CD44, CD200, CEACAM1,CHGA, COL15A1, COL18A1, COL4A1, COL4A2, COL4A3, CSF3, CTGF, CXCL12,CXCL2, DNMT3B, ECGF1, EDG1, EDIL3, ENPP2, EPHB2, FBLN5, F2, FGF1, FGF2,FIGF, FLT4, FN1, FST, FOXC2, GRN, HGF, HEY1, HSPG2, IFNB1, IL8, IL12A,ITGA4, ITGAV, ITGB3, MDK, MMP2, MYOZ2, NRP1, NRP2, PDGFB, PDGFRA,PDGFRB, PECAM1, PF4, PGK1, PROX1, PTN, SEMA3F, SERPINB5, SERPINC1,SERPINF1, TIMP2, TIMP3, TGFA, TGFB1, THBS1, THBS2, TIE1, TIE2/TEK, TNF,TNNI1, TNFSF15, VASH1, VEGF, VEGFB, VEGFC, VEGFR1/FLT1, and/orVEGFR2/KDR; (d) expresses one or more of CD49d, Connexin-43, HLA-ABC,Beta 2-microglobulin, CD349, CD318, PDL1, CD106, Galectin-1, ADAM 17,angiotensinogen precursor, filamin A, alpha-actinin 1, megalin,macrophage acetylated LDL receptor I and II, activin receptor type IIBprecursor, Wnt-9 protein, glial fibrillary acidic protein, astrocyte,myosin-binding protein C, and/or myosin heavy chain, nonmuscle type A;(e) secretes VEGF, HGF, IL-8, MCP-3, FGF2, Follistatin, G-CSF, EGF,ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2, uPAR, and/or Galectin-1,e.g., into culture medium in which the cell grows; (f) expresses microRNAs miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, and/or miR-296 at ahigher level than an equivalent number of bone marrow-derivedmesenchymal stem cells; (g) expresses micro RNAs miR-20a, miR-20b,miR-221, miR-222, miR-15b, and/or miR-16 at a lower level than anequivalent number of bone marrow-derived mesenchymal stem cells; (h)expresses miRNAs miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a,miR-20b, miR-296, miR-221, miR-222, miR-15b, and/or miR-16; and/or (i)expresses increased levels of CD202b, IL-8 and/or VEGF when cultured inless than about 5% O₂, compared to expression of CD202b, IL-8 and/orVEGF under 21% O₂. Further provided herein are populations of cellscomprising AMDACs, e.g. populations of AMDACs, having one or more of theabove-recited characteristics.

In another embodiment, any of the above populations of cells comprisingamnion derived adherent cells secretes angiogenic factors. In specificembodiments, the population of cells secretes vascular endothelialgrowth factor (VEGF), epithelial growth factor (EGF), platelet derivedgrowth factor (PDGF), basic fibroblast growth factor (bFGF), and/orinterleukin-8 (IL-8). In other specific embodiments, the population ofcells comprising amnion-derived angiogenic cells secretes one or moreangiogenic factors and thereby induces human endothelial cells tomigrate in an in vitro wound healing assay. In other specificembodiments, the population of cells comprising amnion derived adherentcells induces maturation, differentiation or proliferation of humanendothelial cells, endothelial progenitors, myocytes or myoblasts.

In another embodiment, any of the above populations of cells comprisingamnion derived adherent cells take up acetylated low density lipoprotein(LDL) when cultured in the presence of extracellular matrix proteins,e.g., collagen type I or IV, and/or one or more angiogenic factors,e.g., VEGF, EGF, PDGF, or bFGF, e.g., on a substrate such as placentalcollagen or MATRIGELTm.

In another embodiment, provided herein is a population of cellscomprising amnion derived adherent cells, wherein said cells areadherent to tissue culture plastic, and wherein said cells are OCT-4⁻,as determined by RT-PCR, and VEGFR2/KDR⁺, CD9⁺, CD54⁺, CD105⁺, CD200⁺,or VE-cadherin⁻, as determined by immunolocalization. In specificembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,98% or 99% of the cells in said population of cells are amnion derivedcells that are OCT-4⁻, as determined by RT-PCR, and VEGFR2/KDR⁺, CD9⁺,CD54⁺, CD105⁺, CD200⁺, or VE-cadherin⁻, as determined byimmunolocalization. In another specific embodiment, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in saidpopulation are amnion derived cells that are OCT-4⁻, as determined byRT-PCR, and VEGFR2/KDR⁺, CD9⁺, CD54⁺, CD105⁺, CD200⁺, and VE-cadherin⁻,as determined by immunolocalization. In another specific embodiment,said cells that are OCT-4⁻, as determined by RT-PCR, and VEGFR2/KDR⁺,CD9⁺, CD54⁺, CD105⁺, CD200⁺, or VE-cadherin⁻, as determined byimmunolocalization, do not express CD34, as detected byimmunolocalization, after exposure to 1 to 100 ng/mL VEGF for 4 to 21days. In another specific embodiment, said cells are also VE-cadherin⁻.

The populations of cells provided herein, comprising amnion derivedadherent cells, are able to form sprouts or tube-like structuresresembling vessels or vasculature. In one embodiment, the populations ofcells comprising amnion derived adherent cells form sprouts or tube-likestructures when cultured in the presence of an angiogenic moiety, e.g.,VEGF, EGF, PDGF or bFGF. In a more specific embodiment, said amnionderived cells that are OCT-4⁻, as determined by RT-PCR, and VEGFR2/KDR⁺,CD9⁺, CD54⁺, CD105⁺, CD200⁺, or VE-cadherin⁻, as determined byimmunolocalization, form sprouts or tube-like structures when saidpopulation of cells is cultured in the presence of vascular endothelialgrowth factor (VEGF).

The amnion derived adherent cells described herein display the abovecharacteristics, e.g., combinations of cell surface markers and/or geneexpression profiles, and/or angiogenic potency and function, in primaryculture, or during proliferation in medium suitable for the culture ofstem cells. Such medium includes, for example, medium comprising 1 to100% DMEM-LG (Gibco), 1 to 100% MCDB-201 (Sigma), 1 to 10% fetal calfserum (FCS) (Hyclone Laboratories), 0.1 to 5×insulin-transferrin-selenium (ITS, Sigma), 0.1 to 5×linolenic-acid-bovine-serum-albumin (LA-BSA, Sigma), 10⁻⁵ to 10⁻¹⁵Mdexamethasone (Sigma), 10⁻² to 10⁻¹⁰ M ascorbic acid 2-phosphate(Sigma), 1 to 50 ng/mL epidermal growth factor (EGF), (R&D Systems), 1to 50 ng/mL platelet derived-growth factor (PDGF-BB) (R&D Systems), and100 U penicillin/1000 U streptomycin. In a specific embodiment, themedium comprises 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma), 2% fetalcalf serum (FCS) (Hyclone Laboratories), 1× insulin-transferrin-selenium(ITS), 1× linolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹Mdexamethasone (Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma),epidermal growth factor (EGF) 10 ng/ml (R&D Systems), plateletderived-growth factor (PDGF-BB) 10 ng/ml (R&D Systems), and 100 Upenicillin/1000 U streptomycin Other suitable media are described below.

The isolated populations of amnion derived adherent cells providedherein can comprise about, at least about, or no more than about, 1×10⁵,5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷ 1×10⁸, 5×10⁸, 1×10⁹ 5×10⁹ 1×10¹⁰,5×10¹⁰, 1×10¹¹ or more amnion derived adherent cells, e.g., in acontainer. In various embodiments, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99% of the cells in the isolated cellpopulations provided herein are amnion derived adherent cells. That is,a population of isolated amnion derived adherent cells can comprise,e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%non-stem cells.

The amnion derived adherent cells provided herein can be cultured on asubstrate. In various embodiments, the substrate can be any surface onwhich culture and/or selection of amnion derived adherent cells, can beaccomplished. Typically, the substrate is plastic, e.g., tissue culturedish or multiwell plate plastic. Tissue culture plastic can be treated,coated or imprinted with a biomolecule or synthetic mimetic agent, e.g.,CELLSTARTTm, MESENCULTTm ACF-substrate, ornithine, or polylysine, or anextracellular matrix protein, e.g., collagen, laminin, fibronectin,vitronectin, or the like.

Amnion derived cells, e.g., the amnion derived adherent cells providedherein, and populations of such cells, can be isolated from one or moreplacentas. For example, an isolated population of the amnion derivedcells provided herein can be a population of placental cells comprisingsuch cells obtained from, or contained within, disrupted amnion tissue,e.g., tissue digestate (that is, the collection of cells obtained byenzymatic digestion of an amnion), wherein said population of cells isenriched for the amnion derived cells, and wherein the tissue is from asingle placenta or from two or more placentas. Isolated amnion derivedcells can be cultured and expanded to produce populations of such cells.Populations of placental cells comprising amnion derived adherent cellscan also be cultured and expanded to produce populations of amnionderived adherent cells.

In certain embodiments, AMDACs displaying any of the above marker and/orgene expression characteristics have been passaged at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, ormore. In certain other embodiments, AMDACs displaying any of the abovemarker and/or gene expression characteristics have been doubled inculture at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or at least50 times, or more.

5.2 Populations of Amnion Derived Adherent Cells Comprising Other CellTypes

The isolated cell populations comprising amnion derived adherent cellsdescribed herein can comprise a second cell type, e.g., placental cellsthat are not amnion derived adherent cells, or, e.g., cells that are notplacental cells. For example, an isolated population of amnion derivedadherent cells can comprise, e.g., can be combined with, a population ofa second type of cells, wherein said second type of cell are, e.g.,embryonic stem cells, blood cells (e.g., placental blood, placentalblood cells, umbilical cord blood, umbilical cord blood cells,peripheral blood, peripheral blood cells, nucleated cells from placentalblood, umbilical cord blood, or peripheral blood, and the like), stemcells isolated from blood (e.g., stem cells isolated from placentalblood, umbilical cord blood or peripheral blood), placental stem cells(e.g., the placental stem cells described in U.S. Pat. No. 7,468,276,and in U.S. Patent Application Publication No. and 2007/0275362, thedisclosures of which are incorporated herein by reference in theirentireties), nucleated cells from placental perfusate, e.g., totalnucleated cells from placental perfusate; umbilical cord stem cells,populations of blood-derived nucleated cells, bone marrow-derivedmesenchymal stromal cells, bone marrow-derived mesenchymal stem cells,bone marrow-derived hematopoietic stem cells, crude bone marrow, adult(somatic) stem cells, populations of stem cells contained within tissue,cultured cells, e.g., cultured stem cells, populations offully-differentiated cells (e.g., chondrocytes, fibroblasts, amnioticcells, osteoblasts, muscle cells, cardiac cells, etc.), pericytes, andthe like. In a specific embodiment, a population of cells comprisingamnion derived adherent cells comprises placental stem cells or stemcells from umbilical cord. In certain embodiments in which the secondtype of cell is blood or blood cells, erythrocytes have been removedfrom the population of cells.

In a specific embodiment, the second type of cell is a hematopoieticstem cell. Such hematopoietic stem cells can be, for example, containedwithin unprocessed placental, umbilical cord blood or peripheral blood;in total nucleated cells from placental blood, umbilical cord blood orperipheral blood; in an isolated population of CD34⁺ cells fromplacental blood, umbilical cord blood or peripheral blood; inunprocessed bone marrow; in total nucleated cells from bone marrow; inan isolated population of CD34⁺ cells from bone marrow, or the like.

In another embodiment, an isolated population of amnion derived adherentcells is combined with a plurality of adult or progenitor cells from thevascular system. In various embodiments, the cells are endothelialcells, endothelial progenitor cells, myocytes, cardiomyocytes,pericytes, angioblasts, myoblasts or cardiomyoblasts.

In a another embodiment, the second cell type is a non-embryonic celltype manipulated in culture in order to express markers of pluripotencyand functions associated with embryonic stem cells

In specific embodiments of the above isolated populations of amnionderived adherent cells, either or both of the amnion derived adherentcells and cells of a second type are autologous, or are allogeneic, toan intended recipient of the cells.

Further provided herein is a composition comprising amnion derivedadherent cells, and a plurality of stem cells other than the amnionderived adherent cells. In a specific embodiment, the compositioncomprises a stem cell that is obtained from a placenta, i.e., aplacental stem cell, e.g., placental stem cells as described in U.S.Pat. Nos. 7,045,148; 7,255,879; and 7,311,905, and in U.S. PatentApplication Publication No. 2007/0275362, the disclosures of each ofwhich are incorporated herein by reference in their entireties. Inspecific embodiments, said placental stem cells are CD200⁺ and HLA-G⁺;CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺;CD73⁺ and CD105⁺ and facilitate the formation of one or moreembryoid-like bodies in a population of placental cells comprising saidstem cell when said population is cultured under conditions that allowthe formation of an embryoid-like body; or OCT-4⁺ and facilitate theformation of one or more embryoid-like bodies in a population ofplacental cells comprising the stem cell when said population iscultured under conditions that allow formation of embryoid-like bodies;or any combination thereof. In a more specific embodiment, said CD200⁺,HLA-G⁺ stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In anothermore specific embodiment, said CD73⁺, CD105⁺, and CD200⁺ stem cells areCD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another more specific embodiment,said CD200⁺, OCT-4⁺ stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺and HLA-G⁺. In another more specific embodiment, said CD73⁺, CD105⁺ andHLA-G⁺ stem cells are CD34⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another morespecific embodiment, said CD73⁺ and CD105⁺ stem cells are OCT-4⁺, CD34⁻,CD38⁻ and CD45⁻. In another more specific embodiment, said OCT-4⁺ stemcells are CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In anothermore specific embodiment, the placental stem cells are maternal inorigin (that is, have the maternal genotype). In another more specificembodiment, the placental stem cells are fetal in origin (that is, havethe fetal genotype).

In another specific embodiment, the composition comprises amnion derivedadherent cells, and embryonic stem cells. In another specificembodiment, the composition comprises amnion derived adherent cells andmesenchymal stromal or stem cells, e.g., bone marrow-derived mesenchymalstromal or stem cells. In another specific embodiment, the compositioncomprises bone marrow-derived hematopoietic stem cells. In anotherspecific embodiment, the composition comprises amnion derived adherentcells and hematopoietic progenitor cells, e.g., hematopoietic progenitorcells from bone marrow, fetal blood, umbilical cord blood, placentalblood, and/or peripheral blood. In another specific embodiment, thecomposition comprises amnion derived adherent cells and somatic stemcells. In a more specific embodiment, said somatic stem cell is a neuralstem cell, a hepatic stem cell, a pancreatic stem cell, an endothelialstem cell, a cardiac stem cell, or a muscle stem cell.

In other specific embodiments, the second type of cells comprise about,at least, or no more than, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50% of cells in said population. In other specific embodiments, theAMDACs in said composition comprise at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85% or 90% of cells in said composition. In other specificembodiments, the amnion derived adherent cells comprise about, at least,or no more than, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of cells insaid population.

Cells in an isolated population of amnion derived adherent cells can becombined with a plurality of cells of another type, e.g., with apopulation of stem cells, in a ratio of about 100,000,000:1,50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1,1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1,10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1,10:1, 5:1, 2:1, 1:1; 1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000;1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000;1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000;1:50,000,000; or about 1:100,000,000, comparing numbers of totalnucleated cells in each population. Cells in an isolated population ofamnion derived adherent cells can be combined with a plurality of cellsof a plurality of cell types, as well.

5.3 Growth in Culture

The growth of the amnion derived adherent cells described herein, as forany mammalian cell, depends in part upon the particular medium selectedfor growth. Under optimum conditions, amnion derived adherent cellstypically double in number in approximately 24 hours. During culture,the amnion derived adherent cells described herein adhere to a substratein culture, e.g. the surface of a tissue culture container (e.g., tissueculture dish plastic, fibronectin-coated plastic, and the like) and forma monolayer. Typically, the cells establish in culture within 2-7 daysafter digestion of the amnion. They proliferate at approximately 0.4 to1.2 population doublings per day and can undergo at least 30 to 50population doublings. The cells display a mesenchymal/fibroblasticcell-like phenotype during subconfluence and expansion, and acuboidal/cobblestone-like appearance at confluence, and proliferation inculture is strongly contact-inhibited. Populations of amnion-derivedangiogenic cells can form embryoid bodies during expansion in culture.

5.4 Methods of Obtaining Amnion-Derived Angiogenic Cells

The amnion derived adherent cells, and populations of cells comprisingthe amnion derived adherent cells, can be produced, e.g., isolated fromother cells or cell populations, for example, through particular methodsof digestion of amnion tissue, optionally followed by assessment of theresulting cells or cell population for the presence or absence ofmarkers, or combinations of markers, characteristics of amnion derivedadherent cells, or by obtaining amnion cells and selecting on the basisof markers characteristic of amnion derived adherent cells.

The amnion derived adherent cells, and isolated populations of cellscomprising the amnion derived adherent cells, provided herein can beproduced by, e.g., digestion of amnion tissue followed by selection foradherent cells. In one embodiment, for instance, isolated amnion derivedadherent cells, or an isolated population of cells comprising amnionderived adherent cells, can be produced by (1) digesting amnion tissuewith a first enzyme to dissociate cells from the epithelial layer of theamnion from cells from the mesenchymal layer of the amnion; (2)subsequently digesting the mesenchymal layer of the amnion with a secondenzyme to form a single-cell suspension; (3) culturing cells in saidsingle-cell suspension on a tissue culture surface, e.g., tissue cultureplastic; and (4) selecting cells that adhere to said surface after achange of medium, thereby producing an isolated population of cellscomprising amnion derived adherent cells. In a specific embodiment, saidfirst enzyme is trypsin. In a more specific embodiment, said trypsin isused at a concentration of 0.25% trypsin (w/v), in 5-20, e.g., 10milliliters solution per gram of amnion tissue to be digested. Inanother more specific embodiment, said digesting with trypsin is allowedto proceed for about 15 minutes at 37° C. and is repeated up to threetimes. In another specific embodiment, said second enzyme iscollagenase. In a more specific embodiment, said collagenase is used ata concentration between 50 and 500 U/L in 5 mL per gram of amnion tissueto be digested. In another more specific embodiment, said digesting withcollagenase is allowed to proceed for about 45-60 minutes at 37° C. Inanother specific embodiment, the single-cell suspension formed afterdigestion with collagenase is filtered through, e.g., a 75 μM-150 μMfilter between step (2) and step (3). In another specific embodiment,said first enzyme is trypsin, and said second enzyme is collagenase.

An isolated population of cells comprising amnion derived adherent cellscan, in another embodiment, be obtained by selecting cells from amnion,e.g., cells obtained by digesting amnion tissue as described elsewhereherein, that display one or more characteristics of an amnion derivedadherent cell. In one embodiment, for example, a cell population isproduced by a method comprising selecting amnion cells that are (a)negative for OCT-4, as determined by RT-PCR, and (b) positive for one ormore of VEGFR2/KDR, CD9, CD54, CD105, CD200, as determined byimmunolocalization; and isolating said cells from other cells to form acell population. In a specific embodiment, said amnion cells areadditionally VE-cadherin⁻. In a specific embodiment, a cell populationis produced by selecting placental cells that are (a) negative forOCT-4, as determined by RT-PCR, and VE-cadherin, as determined byimmunolocalization, and (b) positive for each of VEGFR2/KDR, CD9, CD54,CD105, CD200, as determined by immunolocalization; and isolating saidcells from other cells to form a cell population. In certainembodiments, selection by immunolocalization is performed beforeselection by RT-PCR. In another specific embodiment, said selectingcomprises selecting cells that do not express cellular marker CD34 afterculture for 4 to 21 days in the presence of 1 to 100 ng/mL VEGF.

In another embodiment, for example, a cell population is produced by amethod comprising selecting amnion cells that are adherent to tissueculture plastic and are OCT-4⁻, as determined by RT-PCR, andVEGFR1/Flt-1⁺ and VEGFR2/KDR⁺, as determined by immunolocalization, andisolating said cells from other cells to form a cell population. In aspecific embodiment, a cell population is produced by a methodcomprising selecting amnion cells that are OCT-4⁻, as determined byRT-PCR, and VEGFR1/Flt-1⁺, VEGFR2/KDR⁺, and HLA-G⁻, as determined byimmunolocalization. In another specific embodiment, said cell populationis produced by selecting amnion cells that are additionally one or more,or all, of CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺, Tie-2⁺, TEM-7⁺, CD31⁻,CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻, and/or CXCR4⁻ (chemokine (C-X-Cmotif) receptor 4) as determined by immunolocalization, and isolatingthe cells from cells that do not display one or more of thesecharacteristics. In another specific embodiment, said cell population isproduced by selecting amnion cells that are additionally VE-cadherin⁻ asdetermined by immunolocalization, and isolating the cells from cellsthat are VE-cadherin⁺. In another specific embodiment, said cellpopulation is produced by selecting amnion cells that are additionallyCD105⁺ and CD200⁺ as determined by immunolocalization, and isolating thecells from cells that are CD105⁻ or CD200⁻. In another specificembodiment, said cell does not express CD34 as detected byimmunolocalization after exposure to 1 to 100 ng/mL VEGF for 4 to 21days.

In the selection of cells, it is not necessary to test an entirepopulation of cells for characteristics specific to amnion derivedadherent cells. Instead, one or more aliquots of cells (e.g., about0.5%-2%) of a population of cells may be tested for suchcharacteristics, and the results can be attributed to the remainingcells in the population.

Selected cells can be confirmed to be the amnion derived adherent cellsprovided herein by culturing a sample of the cells (e.g., about 10⁴ toabout 10⁵ cells) on a substrate, e.g., MATRIGEL™, for 4 to 14, e.g., 7,days in the presence of VEGF (e.g., about 50 ng/mL), and visuallyinspecting the cells for the appearance of sprouts and/or cellularnetworks.

Amnion derived adherent cells can be selected by the above markers usingany method known in the art of cell selection. For example, the adherentcells can be selected using an antibody or antibodies to one or morecell surface markers, for example, in immunolocalization, e.g., flowcytometry or FACS. Selection can be accomplished using antibodies inconjunction with magnetic beads. Antibodies that are specific forcertain markers are known in the art and are available commercially,e.g., antibodies to CD9 (Abeam); CD54 (Abeam); CD105 (Abeam; BioDesignInternational, Saco, Me., etc.); CD200 (Abeam) cytokeratin(SigmaAldrich). Antibodies to other markers are also availablecommercially, e.g., CD34, CD38 and CD45 are available from, e.g.,StemCell Technologies or BioDesign International. Primers to OCT-4sequences suitable for RT-PCR can be obtained commercially, e.g., fromMillipore or Invitrogen, or can be readily derived from the humansequence in GenBank Accession No. DQ486513.

Detailed methods of obtaining placenta and amnion tissue, and treatingsuch tissue in order to obtain amnion derived adherent cells, areprovided below.

5.4.1 Cell Collection Composition

Generally, cells can be obtained from amnion from a mammalian placenta,e.g., a human placenta, using a physiologically-acceptable solution,e.g., a cell collection composition. Preferably, the cell collectioncomposition prevents or suppresses apoptosis, prevents or suppressescell death, lysis, decomposition and the like. A cell collectioncomposition is described in detail in related U.S. Patent ApplicationPublication No. 2007/0190042, entitled “Improved Medium for CollectingPlacental Stem Cells and Preserving Organs,” the disclosure of which isincorporated herein by reference in its entirety.

The cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of amnion derived adherent cells, for example, a saline solution(e.g., phosphate-buffered saline, Kreb's solution, modified Kreb'ssolution, Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g.,DMEM, H.DMEM, etc.), and the like, with or without the addition of abuffering component, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES).

The cell collection composition can comprise one or more components thattend to preserve cells, e.g., amnion derived adherent cells, that is,prevent the cells from dying, or delay the death of the cells, reducethe number of cells in a population of cells that die, or the like, fromthe time of collection to the time of culturing. Such components can be,e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNKinhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensivedrug, atrial natriuretic peptide (ANP), adrenocorticotropin,corticotropin-releasing hormone, sodium nitroprusside, hydralazine,adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, aphosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g.,2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate,or clonazepam); a TNF-α inhibitor; and/or an oxygen-carryingperfluorocarbon (e.g., perfluorooctyl bromide, perfluorodecyl bromide,etc.).

The cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE™, hyaluronidase, and the like.The use of cell collection compositions comprising tissue-digestingenzymes is discussed in more detail below.

The cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The cell collection composition can also comprise one or more of thefollowing compounds: adenosine (about 1 mM to about 50 mM); D-glucose(about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50mM); a macromolecule of molecular weight greater than 20,000 daltons, inone embodiment, present in an amount sufficient to maintain endothelialintegrity and cellular viability (e.g., a synthetic or naturallyoccurring colloid, a polysaccharide such as dextran or a polyethyleneglycol present at about 25 g/l to about 100 g/l, or about 40 g/l toabout 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylatedhydroxytoluene, glutathione, vitamin C or vitamin E present at about 25μM to about 100 μM); a reducing agent (e.g., N-acetylcysteine present atabout 0.1 mM to about 5 mM); an agent that prevents calcium entry intocells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/1); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

The amnion derived adherent cells described herein can also becollected, e.g., during and after digestion as described below, into asimple physiologically-acceptable buffer, e.g., phosphate-bufferedsaline, a 0.9% NaCl solution, cell culture medium, or the like.

5.4.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth, or after, e.g., Caesarian section. In a preferredembodiment, the placenta is recovered from a patient after informedconsent and after a complete medical history of the patient is obtainedand is associated with the placenta. Preferably, the medical historycontinues after delivery. Such a medical history can be used tocoordinate subsequent use of the placenta or cells harvested therefrom.For example, human placental cells, e.g., amnion derived adherent cells,can be used, in light of the medical history, for personalized medicinefor the infant, or a close relative, associated with the placenta, orfor parents, siblings, or other relatives of the infant.

Prior to recovery of amnion derived adherent cells, the umbilical cordblood and placental blood are removed. In certain embodiments, afterdelivery, the cord blood in the placenta is recovered. The placenta canbe subjected to a conventional cord blood recovery process. Typically aneedle or cannula is used, with the aid of gravity, to exsanguinate theplacenta (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al.,U.S. Pat. No. 5,415,665). The needle or cannula is usually placed in theumbilical vein and the placenta can be gently massaged to aid indraining cord blood from the placenta. Such cord blood recovery may beperformed commercially, e.g., LifeBank USA, Cedar Knolls, N.J., ViaCord,Cord Blood Registry and Cryocell. Preferably, the placenta is gravitydrained without further manipulation so as to minimize tissue disruptionduring cord blood recovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inU.S. Pat. No. 7,147,626. Preferably, the placenta is delivered to thelaboratory four to twenty-four hours following delivery. In certainembodiments, the proximal umbilical cord is clamped, preferably within4-5 cm (centimeter) of the insertion into the placental disc prior tocord blood recovery. In other embodiments, the proximal umbilical cordis clamped after cord blood recovery but prior to further processing ofthe placenta.

The placenta, prior to cell collection, can be stored under sterileconditions and at a temperature of, e.g., 4 to 25° C. (centigrade),e.g., at room temperature. The placenta may be stored for, e.g., aperiod of for zero to twenty-four hours, up to forty-eight hours, orlonger than forty eight hours, prior to perfusing the placenta to removeany residual cord blood. In one embodiment, the placenta is harvestedfrom between about zero hours to about two hours post-expulsion. Theplacenta can be stored in an anticoagulant solution at a temperature of,e.g., 4 to 25° C. (centigrade). Suitable anticoagulant solutions arewell known in the art. For example, a solution of sodium citrate,heparin or warfarin sodium can be used. In a preferred embodiment, theanticoagulant solution comprises a solution of heparin (e.g., 1% w/w in1:1000 solution). The exsanguinated placenta is preferably stored for nomore than 36 hours before cells are collected.

5.4.3 Physical Disruption and Enzymatic Digestion of Amnion Tissue

In one embodiment, the amnion is separated from the rest of theplacenta, e.g., by blunt dissection, e.g., using the fingers. The amnioncan be dissected, e.g., into parts or tissue segments, prior toenzymatic digestion and adherent cell recovery. Amnion derived adherentcells can be obtained from a whole amnion, or from a small segment ofamnion, e.g., a segment of amnion that is about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900 or about 1000 square millimeters in area.

Amnion derived adherent cells can generally be collected from aplacental amnion or a portion thereof, at any time within about thefirst three days post-expulsion, but preferably between about 0 hoursand 48 hours after expulsion, or about 8 hours and about 18 hourspost-expulsion.

In one embodiment, amnion derived adherent cells are extracted fromamnion tissue by enzymatic digestion using one or more tissue-digestingenzymes. The amnion, or a portion thereof, may, e.g., be digested withone or more enzymes dissolved or mixed into a cell collectioncomposition as described above.

In certain embodiments, the cell collection composition comprises one ormore tissue-disruptive enzyme(s). Enzymatic digestion preferably uses acombination of enzymes, e.g., a combination of a matrix metalloproteaseand a neutral protease, for example, a combination of dispase andcollagenase, e.g., used in sequential order. When more than one proteaseis used, the proteases may be used at the same time to digest the amniontissue, or may be used serially. In one embodiment, for example, theamnion tissue is digested three times with trypsin and then once withcollagenase.

In one embodiment, amnion tissue is enzymatically digested with one ormore of a matrix metalloprotease, a neutral protease, and a mucolyticenzyme. In a specific embodiment, the amnion tissue is digested with acombination of collagenase, dispase, and hyaluronidase. In anotherspecific embodiment, the amnion tissue is digested with a combination ofLIBERASE™ (Boehringer Mannheim Corp., Indianapolis, Ind.) andhyaluronidase. Other enzymes that can be used to disrupt amnion tissueinclude papain, deoxyribonucleases, serine proteases, such as trypsin,chymotrypsin, or elastase. Serine proteases may be inhibited by alpha 2microglobulin in serum and therefore the medium used for digestion can,in certain embodiments, be serum-free. In certain other embodiments,EDTA and DNase are used in the digestion of amnion tissue, e.g., toincrease the efficiency of cell recovery. In certain other embodiments,the digestate is diluted so as to avoid trapping cells within theviscous digest.

Typical concentrations for tissue digestion enzymes include, e.g.,50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for dispase,and 10-100 U/mL for elastase. Proteases can be used in combination, thatis, two or more proteases in the same digestion reaction, or can be usedsequentially in order to isolate amnion derived adherent cells. Forexample, in one embodiment, amnion tissue, or part thereof, is digestedfirst with an appropriate amount of trypsin, at a concentration of about0.25%, for, e.g., 15 minutes, at 37° C., followed by collagenase I atabout 1 to about 2 mg/ml for, e.g., 45 minutes.

In one embodiment, amnion derived adherent cells can be obtained asfollows. The amniotic membrane is cut into segments approximately0.1″×0.1″ to about 5″×5″, e.g., 2″×2″, in size. The epithelial monolayeris removed from the fetal side of the amniotic membrane by tripletrypsinization as follows. The segments of amniotic membrane are placedinto a container with warm (e.g., about 20° C. to about 37° C.)trypsin-EDTA solution (0.25%). The volume of trypsin can range fromabout 5 mL per gram of amnion to about 50 mL per gram of amnion. Thecontainer is agitated for about 5 minutes to about 30 minutes, e.g., 15minutes, while maintaining the temperature constant. The segments ofamniotic membrane are then separated from the trypsin solution by anyappropriate method, such as manually removing the amnion segments, or byfiltration. The trypsinization step can be repeated at least one moretime.

Upon completion of the final trypsinization, the segments of amnioticmembrane are placed back into the container filled with warm trypsinneutralization solution, such as phosphate-buffered saline (PBS)/10%FBS, PBS/5% FBS or PBS/3% FBS. The container is agitated for about 5seconds to about 30 minutes, e.g., 5 minutes. The segments of amnioticmembrane are then separated from the trypsin neutralization solution asdescribed above, and the segments of amniotic membrane are placed intothe container filled with warm PBS, pH 7.2. The container is agitatedfor about 5 seconds to about 30 minutes, and the amniotic membranesegments are then separated from the PBS as described above.

The segments of amniotic membrane are then placed into the containerfilled with warm (e.g., about 20° C. to about 37° C.) digestionsolution. The volume of digestion solution can range from about 5 mL pergram of amnion to about 50 mL per gram of amnion. Digestion solutionscomprise digestion enzymes in an appropriate culture medium, such asDMEM. Typical digestion solutions include collagenase type I (about 50U/mL to about 500 U/mL); collagenase type I (about 50 U/mL to about 500U/mL) plus dispase (about 5 U/mL to about 100 U/mL); and collagenasetype I (about 50 U/mL to about 500 U/mL), dispase (about 2 U/mL to about50 U/mL) and hyaluronidase (about 3 U/mL to about 10 U/mL). Thecontainer is agitated at 37° C. until amnion digestion is substantiallycomplete (approximately 10 minutes to about 90 minutes). Warm PBS/5% FBSis then added to the container at a ratio of about 1 mL per gram ofamniotic tissue to about 50 mL per gram of amniotic tissue. Thecontainer is agitated for about 2 minutes to about 5 minutes. The cellsuspension is then filtered to remove any un-digested tissue using a 40μm to 100 μm filter. The cells are suspended in warm PBS (about 1 mL toabout 500 mL), and then centrifuged at 200×g to about 400×g for about 5minutes to about 30 minutes, e.g. 300×g for about 15 minutes at 20° C.After centrifugation, the supernatant is removed and the cells areresuspended in a suitable culture medium. The cell suspension can befiltered (40 μm to 70 μm filter) to remove any remaining undigestedtissue, yielding a single cell suspension.

In this embodiment, cells in suspension are collected and cultured asdescribed elsewhere herein to produce isolated amnion derived adherentcells, and populations of such cells. The remaining undigested amnion,in this embodiment, can be discarded. The cells released from the amniontissue can be, e.g., collected, e.g., by centrifugation, and cultured instandard cell culture medium.

In any of the digestion protocols herein, the cell suspension obtainedby digestion can be filtered, e.g., through a filter comprising poresfrom about 50 μm to about 150 μm, e.g., from about 75 μm to about 125μm. In a more specific embodiment, the cell suspension can be filteredthrough two or more filters, e.g., a 125 μm filter and a 75 μm filter.

In conjunction with any of the methods described herein, AMDACs can beisolated from the cells released during digestion by selecting cellsthat express one or more characteristics of AMDACs, as described inSection 5.1, above.

AMDACs can also, for example, be isolated using a specific two-stepisolation method comprising digestion with trypsin followed by digestionwith collagenase. Thus, in another aspect, provided herein is a methodof isolating amnion derived adherent cells comprising digesting anamniotic membrane or portion thereof with trypsin such that epithelialcells are released from said amniotic membrane; removing the amnioticmembrane or portion thereof from said epithelial cells; furtherdigesting the amniotic membrane or portion thereof with collagenase suchthat amnion derived adherent cells are released from said amnioticmembrane or portion thereof; and separating said amnion derived adherentcells from said amniotic membrane. In a specific embodiment, digestionof the amniotic membrane or portion thereof is repeated at least once.In another specific embodiment, digestion of the amniotic membrane orportion thereof with collagenase is repeated at least once. In anotherspecific embodiment, the trypsin is at about 0.1%-1.0% (finalconcentration). In a more specific embodiment, the trypsin is at about0.25% (final concentration). In another specific embodiment, thecollagenase is at about 50 U/mL to about 1000 U/mL (finalconcentration). In a more specific embodiment, the collagenase is atabout 125 U/mL (final concentration). In another specific embodiment,the method of isolation additionally comprises culturing said amnionderived adherent cells in cell culture and separating said amnionderived adherent cells from non-adherent cells in said culture toproduce an enriched population of amnion derived adherent cells. In morespecific embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% of cells in said enriched population of amnionderived adherent cells are said amnion derived adherent cells.

In a more specific embodiment of the above methods, the amnion derivedadherent cells are negative for OCT-4, as determined by RT-PCR, and oneor more of HLA-G⁺, CD90⁺, CD105⁺, and CD117⁻, as determined by flowcytometry.

5.4.4 Isolation, Sorting, and Characterization of Amnion DerivedAdherent Cells

Cell pellets can be resuspended in fresh cell collection composition, asdescribed above, or a medium suitable for cell maintenance, e.g.,Dulbecco's Modified Eagle's Medium (DMEM); Iscove's Modified Dulbecco'sMedium (IMDM), e.g. IMDM serum-free medium containing 2 U/mL heparin and2 mM EDTA (GibcoBRL, NY); a mixture of buffer (e.g. PBS, HBSS) with FBS(e.g. 2% v/v); or the like.

Amnion derived adherent cells that have been cultured, e.g., on asurface, e.g., on tissue culture plastic, with or without additionalextracellular matrix coating such as fibronectin, can be passaged orisolated by differential adherence. For example, a cell suspensionobtained from collagenase digestion of amnion tissue, performed asdescribed in Section 5.4.3, above, can be cultured, e.g., for 3-7 daysin culture medium on tissue culture plastic. During culture, a pluralityof cells in the suspension adhere to the culture surface, and, aftercontinued culture, give rise to amnion derived adherent cells.Nonadherent cells, which do not give rise to the amnion derived adherentcells, are removed during medium exchange.

The number and type of cells collected from amnion can be monitored, forexample, by measuring changes in morphology and cell surface markersusing standard cell detection techniques such as immunolocalization,e.g., flow cytometry, cell sorting, immunocytochemistry (e.g., stainingwith tissue specific or cell-marker specific antibodies) fluorescenceactivated cell sorting (FACS), magnetic activated cell sorting (MACS),by examination of the morphology of cells using light or confocalmicroscopy, and/or by measuring changes in gene expression usingtechniques well known in the art, such as PCR and gene expressionprofiling. These techniques can be used, too, to identify cells that arepositive for one or more particular markers. For example, using one ormore antibodies to CD34, one can determine, using the techniques above,whether a cell comprises a detectable amount of CD34; if so, the cell isCD34⁺.

Amnion-derived cells, e.g., cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, can besorted using a fluorescence activated cell sorter (FACS). Fluorescenceactivated cell sorting (FACS) is a well-known method for separatingparticles, including cells, based on the fluorescent properties of theparticles (see, e.g., Kamarch, 1987, Methods Enzymol, 151:150-165).Laser excitation of fluorescent moieties in the individual particlesresults in a small electrical charge allowing electromagnetic separationof positive and negative particles from a mixture. In one embodiment,cell surface marker-specific antibodies or ligands are labeled withdistinct fluorescent labels. Cells are processed through the cellsorter, allowing separation of cells based on their ability to bind tothe antibodies used. FACS sorted particles may be directly depositedinto individual wells of 96-well or 384-well plates to facilitateseparation and cloning.

In one sorting scheme, cells from placenta, e.g., amnion derivedadherent cells, can be sorted on the basis of expression of the markersCD49f, VEGFR2/KDR, and/or Flt-1/VEGFR1. Preferably the cells areidentified as being OCT-4⁻, e.g., by determining the expression of OCT-4by RT-PCR in a sample of the cells, wherein the cells are OCT-4⁻ if thecells in the sample fail to show detectable production of mRNA for OCT-4after 30 cycles. For example, cells from amnion that are VEGFR2/KDR⁺ andVEGFR1/Flt-1⁺ can be sorted from cells that are one or more ofVEGFR2/KDR⁻, and VEGFR1/Flt-1⁺, CD9⁺, CD54⁺, CD105⁺, CD200⁺, and/orVE-cadherin⁻. In a specific embodiment, amnion-derived, tissue cultureplastic-adherent cells that are one or more of CD49f⁻, VEGFR2/KDR⁺,CD9⁺, CD54⁺, CD105⁺, CD200⁺, and/or VE-cadherin⁻, or cells that areVEGFR2/KDR⁺, CD9⁺, CD54⁺, CD105⁺, CD200⁺, and VE-cadherin⁻, are sortedaway from cells not expressing one or more of such marker(s), andselected. In another specific embodiment, CD49f⁻, VEGFR2/KDR⁺,VEGFR1/Flt-1⁺ cells that are additionally one or more, or all, of CD31⁺,CD34⁺, CD45⁺, CD133⁻, and/or Tie-2⁺ are sorted from cells that do notdisplay one or more, or any, of such characteristics. In anotherspecific embodiment, VEGFR2/KDR⁺, VEGFR1/Flt-1⁺ cells that areadditionally one or more, or all, of CD9⁺, CD10⁺, CD44⁺, CD54⁺, CD98⁺,Tie-2⁺, TEM-7⁺, CD31⁻, CD34⁻, CD45⁻, CD133⁻, CD143⁻, CD146⁻, and/orCXCR4⁻, are sorted from cells that do not display one or more, or any,of such characteristics.

Selection for amnion derived adherent cells can be performed on a cellsuspension resulting from digestion, or on isolated cells collected fromdigestate, e.g., by centrifugation or separation using flow cytometry.Selection by expressed markers can be accomplished alone or, e.g., inconnection with procedures to select cells on the basis of theiradherence properties in culture. For example, an adherence selection canbe accomplished before or after sorting on the basis of markerexpression.

With respect to antibody-mediated detection and sorting of placentalcells, any antibody, specific for a particular marker, can be used, incombination with any fluorophore or other label suitable for thedetection and sorting of cells (e.g., fluorescence-activated cellsorting). Antibody/fluorophore combinations to specific markers include,but are not limited to, fluorescein isothiocyanate (FITC) conjugatedmonoclonal antibodies against CD105 (available from R&D Systems Inc.,Minneapolis, Minn.); phycoerythrin (PE) conjugated monoclonal antibodiesagainst CD200 (BD Biosciences Pharmingen); VEGFR2/KDR-Biotin (CD309,Abcam), and the like. Antibodies to any of the markers disclosed hereincan be labeled with any standard label for antibodies that facilitatesdetection of the antibodies, including, e.g., horseradish peroxidase,alkaline phosphatase, β-galactosidase, acetylcholinesterasestreptavidin/biotin, avidin/biotin, umbelliferone, fluorescein,fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin (PE), luminol, luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Amnion derived adherent cells can be labeled with an antibody to asingle marker and detected and/sorted based on the single marker, or canbe simultaneously labeled with multiple antibodies to a plurality ofdifferent markers and sorted based on the plurality of markers.

In another embodiment, magnetic beads can be used to separate cells,e.g., to separate the amnion derived adherent cells described hereinfrom other amnion cells. The cells may be sorted using a magneticactivated cell sorting (MACS) technique, a method for separatingparticles based on their ability to bind magnetic beads (0.5-100 μmdiameter). A variety of useful modifications can be performed on themagnetic microspheres, including covalent addition of antibody thatspecifically recognizes a particular cell surface molecule or hapten.The beads are then mixed with the cells to allow binding. Cells are thenpassed through a magnetic field to separate out cells having thespecific cell surface marker. In one embodiment, these cells can thenisolated and re-mixed with magnetic beads coupled to an antibody againstadditional cell surface markers. The cells are again passed through amagnetic field, isolating cells that bound both the antibodies. Suchcells can then be diluted into separate dishes, such as microtiterdishes for clonal isolation.

Amnion derived adherent cells can be assessed for viability,proliferation potential, and longevity using standard techniques knownin the art, such as trypan blue exclusion assay, fluorescein diacetateuptake assay, propidium iodide uptake assay (to assess viability); andthymidine uptake assay or MTT cell proliferation assay (to assessproliferation). Longevity may be determined by methods well known in theart, such as by determining the maximum number of population doubling inan extended culture.

Amnion derived adherent cells, can also be separated from otherplacental cells using other techniques known in the art, e.g., selectivegrowth of desired cells (positive selection), selective destruction ofunwanted cells (negative selection); separation based upon differentialcell agglutinability in the mixed population as, for example, withsoybean agglutinin; freeze-thaw procedures; filtration; conventional andzonal centrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.5 Culture of Amnion Derived Adherent Cells

5.5.1 Culture Media

Isolated amnion derived adherent cells, or populations of such cells,can be used to initiate, or seed, cell cultures. Cells are generallytransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or biomolecules such as laminin, collagen(e.g., native or denatured), gelatin, fibronectin, ornithine,vitronectin, and extracellular membrane protein (e.g., MATRIGEL™ (BDDiscovery Labware, Bedford, Mass.)).

AMDACs can, for example, be established in media suitable for theculture of stem cells, Establishment media can, for example, includeEGM-2 medium (Lonza), DMEM+10% FBS, or medium comprising 60% DMEM-LG(Gibco), 40% MCDB-201 (Sigma), 2% fetal calf serum (FCS) (HycloneLaboratories), 1× insulin-transferrin-selenium (ITS), 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 Ustreptomycin (referred to herein as “standard medium”).

Amnion derived adherent cells can be cultured in any medium, and underany conditions, recognized in the art as acceptable for the culture ofcells, e.g., adherent placental stem cells. Preferably, the culturemedium comprises serum. In various embodiments, media for the culture orsubculture of AMDACs includes STEMPRO® (Invitrogen), MSCM-sf (ScienCell,Carlsbad, Calif.), MESENCULT®-ACF medium (StemCell Technologies,Vancouver, Canada), standard medium, standard medium lacking EGF,standard medium lacking PDGF, DMEM+10% FBS, EGM-2 (Lonza), EGM-2MV(Lonza), 2%, 10% and 20% ES media, ES-SSR medium, or α-MEM-20% FBS.Medium acceptable for the culture of amnion derived adherent cellsincludes, e.g., DMEM, IMDM, DMEM (high or low glucose), Eagle's basalmedium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove'smodified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGMLonza), ADVANCESTEM™ Medium (Hyclone), KNOCKOUT™ DMEM (Invitrogen),Leibovitz's L-15 medium, MCDB, DMEM/F12, RPMI 1640, advanced DMEM(Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO FREE, or the like. Invarious embodiments, for example, DMEM-LG (Dulbecco's Modified EssentialMedium, low glucose)/MCDB 201 (chick fibroblast basal medium) containingITS (insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serumalbumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1, andpenicillin/streptomycin; DMEM-HG (high glucose) comprising about 2 toabout 20%, e.g., about 10%, fetal bovine serum (FBS; e.g. defined fetalbovine serum, Hyclone, Logan Utah); DMEM-HG comprising about 2 to about20%, e.g., about 15%, FBS; IMDM (Iscove's modified Dulbecco's medium)comprising about 2 to about 20%, e.g., about 10%, FBS, about 2 to about20%, e.g., about 10%, horse serum, and hydrocortisone; M199 comprisingabout 2 to about 20%, e.g., about 10%, FBS, EGF, and heparin; α-MEM(minimal essential medium) comprising about 2 to about 20%, e.g., about10%, FBS, GLUTAMAX™ and gentamicin; DMEM comprising 10% FBS, GLUTAMAX™and gentamicin; DMEM-LG comprising about 2 to about 20%, e.g., about15%, (v/v) fetal bovine serum (e.g., defined fetal bovine serum,Hyclone, Logan Utah), antibiotics/antimycotics (e.g., penicillin atabout 100 Units/milliliter, streptomycin at 100 micrograms/milliliter,and/or amphotericin B at 0.25 micrograms/milliliter (Invitrogen,Carlsbad, Calif.)), and 0.001% (v/v) β-mercaptoethanol (Sigma, St. LouisMo.); KNOCKOUT™-DMEM basal medium supplemented with 2 to 20% FBS,non-essential amino acid (Invitrogen), beta-mercaptoethanol, KNOCKOUT™basal medium supplemented with KNOCKOUT™ Serum Replacement, alpha-MEMcomprising 2 to 20% FBS, EBM2™ basal medium supplemented with EGF, VEGF,bFGF, R3-IGF-1, hydrocortisone, heparin, ascorbic acid, FBS,gentamicin), or the like.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., FCS or FBS, e.g., about 2-20%(v/v); equine (horse) serum (ES); human serum (HS));beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one or moregrowth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

Amnion derived adherent cells (AMDACs) can be cultured in standardtissue culture conditions, e.g., in tissue culture dishes or multiwellplates. The cells can also be cultured using a hanging drop method. Inthis method, the cells are suspended at about 1×10⁴ cells per mL inabout 5 mL of medium, and one or more drops of the medium are placed onthe inside of the lid of a tissue culture container, e.g., a 100 mLPetri dish. The drops can be, e.g., single drops, or multiple dropsfrom, e.g., a multichannel pipetter. The lid is carefully inverted andplaced on top of the bottom of the dish, which contains a volume ofliquid, e.g., sterile PBS sufficient to maintain the moisture content inthe dish atmosphere, and the cells are cultured. AMDACs can also becultured in standard or high-volume or high-throughput culture systems,such as T-flasks, Corning HYPERFLASK®, Cell Factories (Nunc), 1-, 2-,4-, 10 or 40-Tray Cell stacks, and the like.

In one embodiment, amnion derived adherent cells are cultured in thepresence of a compound that acts to maintain an undifferentiatedphenotype in the cells. In a specific embodiment, the compound is asubstituted 3,4-dihydropyridimo[4,5-d]pyrimidine. In a more specificembodiment, the compound is a compound having the following chemicalstructure:

The compound can be contacted with an amnion derived adherent cell, orpopulation of such cells, at a concentration of, for example, betweenabout 1 μM to about 10 μM.

5.5.2 Expansion and Proliferation of Amnion Derived Adherent Cells

Once an isolated amnion derived adherent cell, or isolated population ofsuch cells (e.g., amnion derived adherent cells, or population of suchcells separated from at least 50% of the amnion cells with which thecell or population of cells is normally associated in vivo), the cellscan be proliferated and expanded in vitro. For example, a population ofadherent cells or amnion derived adherent cells can be cultured intissue culture containers, e.g., dishes, flasks, multiwell plates, orthe like, for a sufficient time for the cells to proliferate to 40-70%confluence, that is, until the cells and their progeny occupy 40-70% ofthe culturing surface area of the tissue culture container.

Amnion derived adherent cells can be seeded in culture vessels at adensity that allows cell growth. For example, the cells may be seeded atlow density (e.g., about 400 to about 6,000 cells/cm²) to high density(e.g., about 20,000 or more cells/cm²). In a preferred embodiment, thecells are cultured at about 0% to about 5% by volume CO₂ in air. In somepreferred embodiments, the cells are cultured at about 0.1% to about 25%O₂ in air, preferably about 5% to about 20% O₂ in air. The cells arepreferably cultured at about 25° C. to about 40° C., preferably at about37° C.

The cells are preferably cultured in an incubator. During culture, theculture medium can be static or can be agitated, for example, duringculture using a bioreactor. Amnion derived adherent cells preferably aregrown under low oxidative stress (e.g., with addition of glutathione,ascorbic acid, catalase, tocopherol, N-acetylcysteine, or the like).

Although the amnion-derived angiogenic cells may be grown to confluence,the cells are preferably not grown to confluence. For example, once40%-70% confluence is obtained, the cells may be passaged. For example,the cells can be enzymatically treated, e.g., trypsinized, usingtechniques well-known in the art, to separate them from the tissueculture surface. After removing the cells by pipetting and counting thecells, about 20,000-100,000 cells, preferably about 50,000 cells, orabout 400 to about 6,000 cells/cm², can be passaged to a new culturecontainer containing fresh culture medium. Typically, the new medium isthe same type of medium from which the cells were removed. The amnionderived adherent cells can be passaged at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, or more. AMDACscan be doubled in culture at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or at least 50 times, or more.

5.6 Preservation of Amnion Derived Adherent Cells

Amnion derived adherent cells can be preserved, that is, placed underconditions that allow for long-term storage, or conditions that inhibitcell death by, e.g., apoptosis or necrosis, e.g., during collection orprior to production of the compositions described herein, e.g., usingthe methods described herein.

Amnion derived adherent cells can be preserved using, e.g., acomposition comprising an apoptosis inhibitor, necrosis inhibitor and/oran oxygen-carrying perfluorocarbon, as described in U.S. ApplicationPublication No. 2007/0190042, the disclosure of which is herebyincorporated by reference in its entirety. In one embodiment, a methodof preserving such cells, or a population of such cells, comprisescontacting said cells or population of cells with a cell collectioncomposition comprising an inhibitor of apoptosis and an oxygen-carryingperfluorocarbon, wherein said inhibitor of apoptosis is present in anamount and for a time sufficient to reduce or prevent apoptosis in thepopulation of cells, as compared to a population of cells not contactedwith the inhibitor of apoptosis. In a specific embodiment, saidinhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, said inhibitor of apoptosis is a JNK inhibitor. In a morespecific embodiment, said INK inhibitor does not modulatedifferentiation or proliferation of amnion derived adherent cells. Inanother embodiment, said cell collection composition comprises saidinhibitor of apoptosis and said oxygen-carrying perfluorocarbon inseparate phases. In another embodiment, said cell collection compositioncomprises said inhibitor of apoptosis and said oxygen-carryingperfluorocarbon in an emulsion. In another embodiment, the cellcollection composition additionally comprises an emulsifier, e.g.,lecithin. In another embodiment, said apoptosis inhibitor and saidperfluorocarbon are between about 0° C. and about 25° C. at the time ofcontacting the cells. In another more specific embodiment, saidapoptosis inhibitor and said perfluorocarbon are between about 2° C. and10° C., or between about 2° C. and about 5° C., at the time ofcontacting the cells. In another more specific embodiment, saidcontacting is performed during transport of said population of cells. Inanother more specific embodiment, said contacting is performed duringfreezing and thawing of said population of cells.

Populations of amnion derived adherent cells can be preserved, e.g., bya method comprising contacting a population of said cells with aninhibitor of apoptosis and an organ-preserving compound, wherein saidinhibitor of apoptosis is present in an amount and for a time sufficientto reduce or prevent apoptosis in the population of cells, as comparedto a population of cells not contacted with the inhibitor of apoptosis.In a specific embodiment, the organ-preserving compound is UW solution(described in U.S. Pat. No. 4,798,824; also known as ViaSpan; see alsoSouthard et al., Transplantation 49(2):251-257 (1990)) or a solutiondescribed in Stern et al., U.S. Pat. No. 5,552,267. In anotherembodiment, said organ-preserving compound is hydroxyethyl starch,lactobionic acid, raffinose, or a combination thereof. In anotherembodiment, the cell collection composition additionally comprises anoxygen-carrying perfluorocarbon, either in two phases or as an emulsion.

In another embodiment of the method, amnion derived adherent cells arecontacted with a cell collection composition comprising an apoptosisinhibitor and oxygen-carrying perfluorocarbon, organ-preservingcompound, or combination thereof, during perfusion. In anotherembodiment, the amnion derived adherent cells are contacted with such acell collection composition during a process of tissue disruption, e.g.,enzymatic digestion of amnion tissue. In another embodiment, amnionderived adherent cells are contacted with said cell collection compoundafter collection by tissue disruption, e.g., enzymatic digestion ofamnion tissue.

Typically, during collection of amnion derived adherent cells,enrichment and isolation, it is preferable to minimize or eliminate cellstress due to hypoxia and mechanical stress. In another embodiment ofthe method, therefore, an amnion derived adherent cell, or population ofcells comprising the amnion derived adherent cells, is exposed to ahypoxic condition during collection, enrichment or isolation for lessthan six hours during said preservation, wherein a hypoxic condition isa concentration of oxygen that is, e.g., less than normal atmosphericoxygen concentration; less than normal blood oxygen concentration; orthe like. In a more specific embodiment, said cells or population ofsaid cells is exposed to said hypoxic condition for less than two hoursduring said preservation. In another more specific embodiment, saidcells or population of said cells is exposed to said hypoxic conditionfor less than one hour, or less than thirty minutes, or is not exposedto a hypoxic condition, during collection, enrichment or isolation. Inanother specific embodiment, said population of cells is not exposed toshear stress during collection, enrichment or isolation.

Amnion derived adherent cells can be cryopreserved, in general or by thespecific methods disclosed herein, e.g., in cryopreservation medium insmall containers, e.g., ampoules. Suitable cryopreservation mediumincludes, but is not limited to, culture medium including, e.g., growthmedium, or cell freezing medium, for example commercially available cellfreezing medium, e.g., cell freezing medium identified by SigmaAldrichcatalog numbers C2695, C2639 (Cell Freezing Medium-Serum-free 1×, notcontaining DMSO) or C6039 (Cell Freezing Medium-Glycoerol 1× containingMinimum Essential Medium, glycerol, calf serum and bovine serum), LonzaPROFREEZE™ 2× Medium, methylcellulose, dextran, human serum albumin,fetal bovine serum, fetal calf serum, or Plasmalyte. Cryopreservationmedium preferably comprises DMSO (dimethylsulfoxide) or glycerol, at aconcentration of, e.g., about 1% to about 20%, e.g., about 5% to 10%(v/v), optionally including fetal bovine serum or human serum.Cryopreservation medium may comprise additional agents, for example,methylcellulose and/or glycerol. Isolated amnion derived adherent cellsare preferably cooled at about 1° C./min during cryopreservation. Apreferred cryopreservation temperature is about −80° C. to about −180°C., preferably about −125° C. to about −140° C. Cryopreserved cells canbe transferred to vapor phase of liquid nitrogen prior to thawing foruse. In some embodiments, for example, once the ampoules have reachedabout −80° C., they are transferred to a liquid nitrogen storage area.Cryopreservation can also be done using a controlled-rate freezer.Cryopreserved cells preferably are thawed at a temperature of about 25°C. to about 40° C., preferably to a temperature of about 37° C.

5.7 Production of a Bank of Amnion Derived Adherent Cells

Amnion derived adherent cells can be cultured in a number of differentways to produce a set of lots, e.g., a set of individually-administrabledoses, of such cells. Sets of lots of angiogenic amniotic cells,obtained from a plurality of placentas, can be arranged in a bank ofcells for, e.g., long-term storage. Generally, amnion derived adherentcells are obtained from an initial culture of cells to form a seedculture, which is expanded under controlled conditions to formpopulations of cells from approximately equivalent numbers of doublings.Lots are preferably derived from the tissue of a single placenta, butcan be derived from the tissue of a plurality of placentas.

In one non-limiting embodiment, lots or doses of amnion derived adherentcells are obtained as follows. Amnion tissue is first disrupted, e.g.,digested as described in Section 5.4.3, above using serial trypsin andcollagenase digestions. Cells from the collagenase-digested tissue arecultured, e.g., for about 1-3 weeks, preferably about 2 weeks. Afterremoval of non-adherent cells, high-density colonies that form arecollected, e.g., by trypsinization. These cells are collected andresuspended in a convenient volume of culture medium, and defined asPassage 0 cells.

Passage 0 cells can then be used to seed expansion cultures. Expansioncultures can be any arrangement of separate cell culture apparatuses,e.g., a Cell Factory by NUNC™ Cells in the Passage 0 culture can besubdivided to any degree so as to seed expansion cultures with, e.g.,1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, or 10×10⁴adherent cells. Preferably, from about 1×10³ to about 3×10⁴ Passage 0cells are used to seed each expansion culture. The number of expansioncultures can depend upon the number of Passage 0 cells, and may begreater or fewer in number depending upon the particular placenta(s)from which the adherent cells are obtained.

Expansion cultures can then be grown until the density of cells inculture reaches a certain value, e.g., about 1×10⁵ cells/cm². Cells caneither be collected and cryopreserved at this point, or passaged intonew expansion cultures as described above. Cells can be passaged, e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20times prior to use. A record of the cumulative number of populationdoublings is preferably maintained during expansion culture(s). Thecells from a Passage 0 culture can be expanded for 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40doublings, or up to 60 doublings. Preferably, however, the number ofpopulation doublings, prior to dividing the population of cells intoindividual doses, is between about 15 and about 30 doublings. The cellscan be culture continuously throughout the expansion process, or can befrozen at one or more points during expansion.

Cells to be used for individual doses can be frozen, e.g., cryopreservedfor later use. Individual doses can comprise, e.g., about 1 million toabout 50 million cells per mL, and can comprise between about 10⁶ andabout 10¹⁰ cells in total.

In one embodiment, therefore, a cell bank comprising amnion derivedadherent cells can be made by a method comprising: expanding primaryculture amnion derived adherent cells from a human post-partum placentafor a first plurality of population doublings; cryopreserving the cellsto form a Master Cell Bank; optionally expanding a plurality of thecells from the Master Cell Bank for a second plurality of populationdoublings; cryopreserving the expanded cells to form a Working CellBank; optionally expanding a plurality of the expanded amnion derivedadherent cells from the Working Cell Bank for a third plurality ofpopulation doublings; and cryopreserving the resulting expanded cells inindividual doses, wherein said individual doses collectively compose acell bank. The bank can comprise doses, or lots, of solely amnionderived adherent cells, or can comprise a combination of lots of amnionderived adherent cells and lots or doses of another kind of cell, e.g.,another kind of stem or progenitor cell. Preferably, each individualdose comprises only amnion derived adherent cells. In another specificembodiment, all of said cells in said primary culture are from the sameplacenta. In another specific embodiment, said individual doses comprisefrom about 10⁴ to about 10⁵ cells. In another specific embodiment, saidindividual doses comprise from about 10⁵ to about 10⁶ cells. In anotherspecific embodiment, said individual doses comprise from about 10⁶ toabout 10⁷ cells. In another specific embodiment, said individual dosescomprise from about 10⁷ to about 10⁸ cells. In another specificembodiment, said individual doses comprise from about 10⁸ to about 10⁹cells. In another specific embodiment, said individual doses comprisefrom about 10⁹ to about 10¹⁰ cells.

In certain embodiments, amnion derived adherent cells can be thawed froma Working Cell Bank and cultured for a plurality of populationdoublings. When a desired number of cells is generated, or a desirednumber of population doublings has taken place, the adherent cells canbe collected, e.g., by centrifugation, and resuspended in a solutioncomprising, e.g., dextran, e.g., 5% dextran. In certain embodiments, thedextran is dextran-40. In certain embodiments, the cells are collected asecond time and resuspended in a solution comprising dextran and acryopreservant, e.g., a 5% dextran (e.g., dextran-40) solutioncomprising 10% HSA and 2%-20%, e.g., 5% DMSO, and cryopreserved. Thecryopreserved amnion derived adherent cells can be thawed, e.g.,immediately before use.

In a preferred embodiment, the donor from which the placenta is obtained(e.g., the mother) is tested for at least one pathogen. In certainembodiments, if the mother tests positive for a tested pathogen, theentire lot from the placenta is discarded. Such testing can be performedat any time during production of lots of amnion derived adherent cells,including before or after establishment of Passage 0 cells, or duringexpansion culture. Pathogens for which the presence is tested caninclude, without limitation, hepatitis A, hepatitis B, hepatitis C,hepatitis D, hepatitis E, human immunodeficiency virus (types I and II),cytomegalovirus, herpesvirus, and the like.

5.8 Uses of Amnion Derived Adherent Cells

Provided herein are compositions comprising amnion derived adherentcells. Examples of such compositions include pharmaceutical compositions(see Section 5.8.1, below); matrices and scaffolds (see Section 5.8.2,below), and media conditioned by amnion derived adherent cells (seeSection 5.8.3, below).

5.8.1 Compositions Comprising Amnion Derived Adherent Cells

In certain embodiments, amnion derived adherent cells are containedwithin, or are components of, a pharmaceutical composition. The cellscan be prepared in a form that is easily administrable to an individual,e.g., amnion derived adherent cells that are contained within acontainer that is suitable for medical use. Such a container can be, forexample, a syringe, sterile plastic bag, flask, jar, or other containerfrom which the anion derived angiogenic cell population can be easilydispensed. For example, the container can be a blood bag or otherplastic, medically-acceptable bag suitable for the intravenousadministration of a liquid to a recipient. The container, in certainembodiments, is one that allows for cryopreservation of the cells. Thecells in the compositions, e.g., pharmaceutical compositions, providedherein, can comprise amnion derived adherent cells derived from a singledonor, or from multiple donors. The cells can be completely HLA-matchedto an intended recipient, or partially or completely HLA-mismatched.

Thus, in one embodiment, amnion derived adherent cells in thecompositions provided herein are administered to an individual in needthereof in the form of a composition comprising amnion derived adherentcells in a container. In another specific embodiment, the container is abag, flask, or jar. In more specific embodiment, said bag is a sterileplastic bag. In a more specific embodiment, said bag is suitable for,allows or facilitates intravenous administration of said adherent cells,e.g., by intravenous infusion, bolus injection, or the like. The bag cancomprise multiple lumens or compartments that are interconnected toallow mixing of the cells and one or more other solutions, e.g., a drug,prior to, or during, administration. In another specific embodiment,prior to cryopreservation, the solution comprising the amnion derivedadherent cells comprises one or more compounds that facilitatecryopreservation of the cells. In another specific embodiment, saidamnion derived adherent cells are contained within aphysiologically-acceptable aqueous solution. In a more specificembodiment, said physiologically-acceptable aqueous solution is a 0.9%NaCl solution. In another specific embodiment, said amnion derivedadherent cells comprise placental cells that are HLA-matched to arecipient of said cells. In another specific embodiment, said amnionderived adherent cells comprise cells that are at least partiallyHLA-mismatched to a recipient of said cells. In another specificembodiment, said amnion derived adherent cells are derived from aplurality of donors. In various specific embodiments, said containercomprises about, at least, or at most 1×10⁶ said cells, 5×10⁶ saidcells, 1×10⁷ said stem cells, 5×10⁷ said cells, 1×10⁸ said cells, 5×10⁸said cells, 1×10⁹ said cells, 5×10⁹ said cells, or 1×10¹⁰ said cells. Inother specific embodiments of any of the foregoing cryopreservedpopulations, said cells have been passaged about, at least, or no morethan 5 times, no more than 10 times, no more than 15 times, or no morethan 20 times. In another specific embodiment of any of the foregoingcryopreserved cells, said cells have been expanded within saidcontainer. In specific embodiments, a single unit dose of amnion derivedadherent cells can comprise, in various embodiments, about, at least, orno more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or more amnion derived adherentcells.

In certain embodiments, the pharmaceutical compositions provided hereincomprises populations of amnion derived adherent cells, that comprise50% viable cells or more (that is, at least 50% of the cells in thepopulation are functional or living). Preferably, at least 60% of thecells in the population are viable. More preferably, at least 70%, 80%,90%, 95%, or 99% of the cells in the population in the pharmaceuticalcomposition are viable.

5.8.2 Matrices Comprising Amnion Derived Adherent Cells

Further provided herein are compositions comprising matrices, hydrogels,scaffolds, and the like. Such compositions can be used in the place of,or in addition to, such cells in liquid suspension.

The matrix can be, e.g., a permanent or degradable decellularizedtissue, e.g., a decellularized amniotic membrane, or a synthetic matrix.The matrix can be a three-dimensional scaffold. In a more specificembodiment, said matrix comprises collagen, gelatin, laminin,fibronectin, pectin, ornithine, or vitronectin. In another more specificembodiment, the matrix is an amniotic membrane or an amnioticmembrane-derived biomaterial. In another more specific embodiment, saidmatrix comprises an extracellular membrane protein. In another morespecific embodiment, said matrix comprises a synthetic compound. Inanother more specific embodiment, said matrix comprises a bioactivecompound. In another more specific embodiment, said bioactive compoundis a growth factor, a cytokine, an antibody, or an organic molecule ofless than 5,000 daltons.

The amnion derived adherent cells described herein can be seeded onto anatural matrix, e.g., a placental biomaterial such as an amnioticmembrane material. Such an amniotic membrane material can be, e.g.,amniotic membrane dissected directly from a mammalian placenta; fixed orheat-treated amniotic membrane, substantially dry (i.e., <20% H₂O)amniotic membrane, chorionic membrane, substantially dry chorionicmembrane, substantially dry amniotic and chorionic membrane, and thelike. Preferred placental biomaterials on which the amnion derivedadherent cells provided herein can be seeded are described in Hariri,U.S. Application Publication No. 2004/0048796, the disclosure of whichis incorporated by reference herein in its entirety.

In another specific embodiment, the matrix is a composition comprisingan extracellular matrix. In a more specific embodiment, said compositionis MATRIGEL™ (BD Biosciences).

The isolated amnion derived adherent cells described herein can besuspended in a hydrogel solution suitable for, e.g., injection. Thehydrogel is, e.g., an organic polymer (natural or synthetic) that iscross-linked via covalent, ionic, or hydrogen bonds to create athree-dimensional open-lattice structure that entraps water molecules toform a gel. Suitable hydrogels for such compositions includeself-assembling peptides, such as RAD16. In one embodiment, a hydrogelsolution comprising the cells can be allowed to harden, for instance ina mold, to form a matrix having cells dispersed therein forimplantation. The amnion derived adherent cells in such a matrix canalso be cultured so that the cells are mitotically expanded, e.g., priorto implantation. Hydrogel-forming materials include polysaccharides suchas alginate and salts thereof, peptides, polyphosphazines, andpolyacrylates, which are crosslinked ionically, or block polymers suchas polyethylene oxide-polypropylene glycol block copolymers which arecrosslinked by temperature or pH, respectively. In some embodiments, thehydrogel or matrix is biodegradable.

In certain embodiments, the compositions comprising cells, providedherein, comprise an in situ polymerizable gel (see., e.g., U.S. PatentApplication Publication 2002/0022676; Anseth et al., J. Control Release,78(1-3):199-209 (2002); Wang et al., Biomaterials, 24(22):3969-80(2003). In some embodiments, the polymers are at least partially solublein aqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

In a specific embodiment, the matrix is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another preferred embodimentthe cells of the invention are seeded onto foam scaffolds that may becomposite structures. In addition, the three-dimensional framework maybe molded into a useful shape, such as a specific structure in the bodyto be repaired, replaced, or augmented. Other examples of scaffolds thatcan be used include nonwoven mats, porous foams, or self assemblingpeptides. Nonwoven mats can be formed using fibers comprised of asynthetic absorbable copolymer of glycolic and lactic acids (e.g.,PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.). Foams, composed of,e.g., poly(ε-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer,formed by processes such as freeze-drying, or lyophilization (see, e.g.,U.S. Pat. No. 6,355,699), can also be used as scaffolds.

The amnion derived adherent cells described herein can be seeded onto athree-dimensional framework or scaffold and implanted in vivo. Such aframework can be implanted in combination with any one or more growthfactors, cells, drugs or other components that, e.g., stimulate tissueformation, e.g., bone formation or formation of vasculature.

The placental amnion derived adherent cells provided herein can, inanother embodiment, be seeded onto foam scaffolds that may be compositestructures. Such foam scaffolds can be molded into a useful shape, suchas that of a portion of a specific structure in the body to be repaired,replaced or augmented. In some embodiments, the framework is treated,e.g., with 0.1M acetic acid followed by incubation in polylysine, PBS,and/or collagen, prior to inoculation of the cells in order to enhancecell attachment. External surfaces of a matrix may be modified toimprove the attachment or growth of cells and differentiation of tissue,such as by plasma-coating the matrix, or addition of one or moreproteins (e.g., collagens, elastic fibers, reticular fibers),glycoproteins, glycosaminoglycans (e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratinsulfate, etc.), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, and plant gums, andthe like.

In some embodiments, the matrix comprises, or is treated with, materialsthat render it non-thrombogenic. These treatments and materials may alsopromote and sustain endothelial growth, migration, and extracellularmatrix deposition. Examples of these materials and treatments includebut are not limited to natural materials such as basement membraneproteins such as laminin and Type IV collagen, synthetic materials suchas EPTFE, and segmented polyurethaneurea silicones, such as PURSPAN™(The Polymer Technology Group, Inc., Berkeley, Calif.). The matrix canalso comprise anti-thrombotic agents such as heparin; the scaffolds canalso be treated to alter the surface charge (e.g., coating with plasma)prior to seeding with the adherent cells provided herein.

The framework may be treated prior to inoculation of the amnion derivedadherent cells provided herein in order to enhance cell attachment. Forexample, prior to inoculation with the cells of the invention, nylonmatrices could be treated with 0.1 molar acetic acid and incubated inpolylysine, PBS, and/or collagen to coat the nylon. Polystyrene can besimilarly treated using sulfuric acid.

In addition, the external surfaces of the three-dimensional frameworkmay be modified to improve the attachment or growth of cells anddifferentiation of tissue, such as by plasma coating the framework oraddition of one or more proteins (e.g., collagens, elastic fibers,reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparinsulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,keratin sulfate), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, or plant gums.

In some embodiments, the matrix comprises or is treated with materialsthat render the matrix non-thrombogenic, e.g., natural materials such asbasement membrane proteins such as laminin and Type IV collagen, andsynthetic materials such as ePTFE or segmented polyurethaneureasilicones, such as PURSPAN (The Polymer Technology Group, Inc.,Berkeley, Calif.). Such materials can be further treated to render thescaffold non-thrombogenic, e.g., with heparin, and treatments that alterthe surface charge of the material, such as plasma coating.

The therapeutic cell compositions comprising amnion derived adherentcells can also be provided in the form of a matrix-cell complex.Matrices can include biocompatible scaffolds, lattices, self-assemblingstructures and the like, whether bioabsorbable or not, liquid, gel, orsolid. Such matrices are known in the arts of therapeutic celltreatment, surgical repair, tissue engineering, and wound healing. Incertain embodiments, the cells adhere to the matrix. In otherembodiments, the cells are entrapped or contained within matrix spaces.Most preferred are those matrix-cell complexes in which the cells growin close association with the matrix and when used therapeutically,stimulate and support ingrowth of a recipient's cells, or stimulate orsupport angiogenesis. The matrix-cell compositions can be introducedinto an individual's body in any way known in the art, including but notlimited to implantation, injection, surgical attachment, transplantationwith other tissue, injection, and the like. In some embodiments, thematrices form in vivo, or in situ. For example, in situ polymerizablegels can be used in accordance with the invention. Examples of such gelsare known in the art.

In some embodiments, the cells provided herein are seeded onto suchthree-dimensional matrices, such as scaffolds and implanted in vivo,where the seeded cells may proliferate on or in the framework or helpestablish replacement tissue in vivo with or without cooperation ofother cells. Growth of the amnion derived adherent cells or co-culturesthereof on the three-dimensional framework preferably results in theformation of a three-dimensional tissue, or foundation thereof, whichcan be utilized in vivo, for example for repair of damaged or diseasedtissue. For example, the three-dimensional scaffolds can be used to formtubular structures, for example for use in repair of blood vessels; oraspects of the circulatory system or coronary structures. In accordancewith one aspect of the invention, amnion derived adherent cells, orco-cultures thereof, are inoculated, or seeded on a three-dimensionalframework or matrix, such as a scaffold, a foam or hydrogel. Theframework may be configured into various shapes such as generally flat,generally cylindrical or tubular, or can be completely free-form as maybe required or desired for the corrective structure under consideration.In some embodiments, the amnion derived adherent cells grow on the threedimensional structure, while in other embodiments, the cells onlysurvive, or even die, but stimulate or promote ingrowth of new tissue orvascularization in a recipient.

The cells of the invention can be grown freely in culture, removed fromthe culture and inoculated onto a three-dimensional framework.Inoculation of the three-dimensional framework with a concentration ofcells, e.g., approximately 10⁶ to 5×10⁷ cells per milliliter, preferablyresults in the establishment of the three-dimensional support inrelatively shorter periods of time. Moreover in some application it maybe preferably to use a greater or lesser number of cells depending onthe result desired.

In a specific embodiment, the matrix can be cut into a strip (e.g.,rectangular in shape) of which the width is approximately equal to theinner circumference of a tubular organ into which it will ultimately beinserted. The amnion derived adherent cells can be inoculated onto thescaffold and incubated by floating or suspending in liquid media. At theappropriate stage of confluence, the scaffold can be rolled up into atube by joining the long edges together. The seam can then be closed bysuturing the two edges together using fibers of a suitable material ofan appropriate diameter. In order to prevent cells from occluding thelumen, one of the open ends of the tubular framework can be affixed to anozzle. Liquid media can be forced through the nozzle from a sourcechamber connected to the incubation chamber to create a current throughthe interior of the tubular framework. The other open end can be affixedto an outflow aperture which leads into a collection chamber from whichthe media can be recirculated through the source chamber. The tube canbe detached from the nozzle and outflow aperture when incubation iscomplete. See, e.g., International Application No. WO 94/25584.

In general, two three-dimensional frameworks can be combined into a tubein accordance with the invention using any of the following methods. Twoor more flat frameworks can be laid atop another and sutured together.The resulting two-layer sheet can then be rolled up, and, as describedabove, joined together and secured. In certain embodiments, one tubularscaffold that is to serve as the inner layer can be inoculated withamnion derived adherent cells and incubated. A second scaffold can begrown as a flat strip with width slightly larger than the outercircumference of the tubular framework. After appropriate growth isattained, the flat framework is wrapped around the outside of thetubular scaffold followed by closure of the seam of the two edges of theflat framework and securing the flat framework to the inner tube. Inanother embodiment, two or more tubular meshes of slightly differingdiameters can be grown separately. The framework with the smallerdiameter can be inserted inside the larger one and secured. For each ofthese methods, more layers can be added by reapplying the method to thedouble-layered tube. The scaffolds can be combined at any stage ofgrowth of the amnion derived adherent cells, and incubation of thecombined scaffolds can be continued when desirable.

In conjunction with the above, the cells and therapeutic compositionsprovided herein can be used in conjunction with implantable devices. Forexample the amnion derived adherent cells can be coadminstered with, forexample, stents, artificial valves, ventricular assist devices,Guglielmi detachable coils and the like. As the devices may constitutethe dominant therapy provided to an individual in need of such therapy,the cells and the like may be used as supportive or secondary therapy toassist in, stimulate, or promote proper healing in the area of theimplanted device. The cells and therapeutic compositions of theinvention may also be used to pretreat certain implantable devices, tominimize problems when they are used in vivo. Such pretreated devices,including coated devices, may be better tolerated by patients receivingthem, with decrease risk of local or systemic infection, or for example,restenosis or further occlusion of blood vessels.

5.8.3 Media Conditioned by Amnion Derived Adherent Cells

Further provided herein is medium that has been conditioned by amnionderived adherent cells, that is, medium comprising one or morebiomolecules secreted or excreted by the adherent cells. In variousembodiments, the conditioned medium comprises medium in which the cellshave grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ormore days, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 population doublings, or more. In otherembodiments, the conditioned medium comprises medium in which amnionderived adherent cells have grown to at least 30%, 40%, 50%, 60%, 70%,80%, 90% confluence, or up to 100% confluence. Such conditioned mediumcan be used to support the culture of a population of cells, e.g., stemcells, e.g., placental stem cells, embryonic stem cells, embryonic germcells, adult stem cells, or the like. In another embodiment, theconditioned medium comprises medium in which amnion derived adherentcells, and cells that are not amnion derived adherent cells, have beencultured together.

The conditioned medium can comprise the adherent cells provided herein.Thus, provided herein is a cell culture comprising amnion derivedadherent cells. In a specific embodiment, the conditioned mediumcomprises a plurality, e.g., a population, of amnion derived adherentcells.

5.9 Modified Amnion Derived Adherent Cells

5.9.1 Genetically Modified Amnion Derived Adherent Cells

In another aspect, the amnion derived adherent cells described hereincan be genetically modified, e.g., to produce a nucleic acid orpolypeptide of interest, or to produce a differentiated cell, e.g., anosteogenic cell, myocytic cell, pericytic cell, or angiogenic cell, thatproduces a nucleic acid or polypeptide of interest. For example, theamnion derived adherent cells can be modified to produce angiogenicfactors, such as proangiogenic molecules, soluble factors and receptorsor promigratory molecules such as chemokines, e.g., stromal cell derivedfactor 1 (SDF-1) or chemokine receptors. Genetic modification can beaccomplished, e.g., using virus-based vectors including, but not limitedto, non-integrating replicating vectors, e.g., papilloma virus vectors,SV40 vectors, adenoviral vectors; integrating viral vectors, e.g.,retrovirus vector or adeno-associated viral vectors; orreplication-defective viral vectors. Other methods of introducing DNAinto cells include the use of liposomes, electroporation, a particlegun, direct DNA injection, or the like.

The adherent cells provided herein can be, e.g., transformed ortransfected with DNA controlled by or in operative association with, oneor more appropriate expression control elements, for example, promoteror enhancer sequences, transcription terminators, polyadenylation sites,internal ribosomal entry sites. Preferably, such a DNA incorporates aselectable marker. Following the introduction of the foreign DNA,engineered adherent cells can be, e.g., grown in enriched media and thenswitched to selective media. In one embodiment, the DNA used to engineera amnion derived adherent cell comprises a nucleotide sequence encodinga polypeptide of interest, e.g., a cytokine, growth factor,differentiation agent, or therapeutic polypeptide.

The DNA used to engineer the adherent cell can comprise any promoterknown in the art to drive expression of a nucleotide sequence inmammalian cells, e.g., human cells. For example, promoters include, butare not limited to, CMV promoter/enhancer, SV40 promoter, papillomaviruspromoter, Epstein-Barr virus promoter, elastin gene promoter, and thelike. In a specific embodiment, the promoter is regulatable so that thenucleotide sequence is expressed only when desired. Promoters can beeither inducible (e.g., those associated with metallothionein and heatshock proteins) or constitutive.

In another specific embodiment, the promoter is tissue-specific orexhibits tissue specificity. Examples of such promoters include but arenot limited to myosin light chain-2 gene control region (Shani, 1985,Nature 314:283) (skeletal muscle).

The amnion derived adherent cells disclosed herein may be engineered orotherwise selected to “knock out” or “knock down” expression of one ormore genes in such cells. The expression of a gene native to a cell canbe diminished by, for example, inhibition of expression by inactivatingthe gene completely by, e.g., homologous recombination. In oneembodiment, for example, an exon encoding an important region of theprotein, or an exon 5′ to that region, is interrupted by a positiveselectable marker, e.g., neo, preventing the production of normal mRNAfrom the target gene and resulting in inactivation of the gene. A genemay also be inactivated by creating a deletion in part of a gene or bydeleting the entire gene. By using a construct with two regions ofhomology to the target gene that are far apart in the genome, thesequences intervening the two regions can be deleted (Mombaerts et al.,1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084). Antisense, morpholinos,DNAzymes, small interfering RNA, short hairpin RNA, and ribozymemolecules that inhibit expression of the target gene can also be used toreduce the level of target gene activity in the adherent cells. Forexample, antisense RNA molecules which inhibit the expression of majorhistocompatibility gene complexes (HLA) have been shown to be mostversatile with respect to immune responses. Triple helix molecules canbe utilized in reducing the level of target gene activity. See, e.g., L.G. Davis et al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nded., Appleton & Lange, Norwalk, Conn., which is incorporated herein byreference.

In a specific embodiment, the amnion derived adherent cells disclosedherein can be genetically modified with a nucleic acid moleculecomprising a nucleotide sequence encoding a polypeptide of interest,wherein expression of the polypeptide of interest is controllable by anexogenous factor, e.g., polypeptide, small organic molecule, or thelike. The polypeptide of interest can be a therapeutic polypeptide. In amore specific embodiment, the polypeptide of interest is IL-12 orinterleukin-1 receptor antagonist (IL-1Ra). In another more specificembodiment, the polypeptide of interest is a fusion of interleukin-1receptor antagonist and dihydrofolate reductase (DHFR), and theexogenous factor is an antifolate, e.g., methotrexate. Such a constructis useful in the engineering of amnion derived adherent cells thatexpress IL-1Ra, or a fusion of IL-1Ra and DHFR, upon contact withmethotrexate. Such a construct can be used, e.g., in the treatment ofrheumatoid arthritis. In this embodiment, the fusion of IL-1Ra and DHFRis translationally upregulated upon exposure to an antifolate such asmethotrexate. Therefore, in another specific embodiment, the nucleicacid used to genetically engineer an amnion derived adherent cell cancomprise nucleotide sequences encoding a first polypeptide and a secondpolypeptide, wherein said first and second polypeptides are expressed asa fusion protein that is translationally upregulated in the presence ofan exogenous factor. The polypeptide can be expressed transiently orlong-term (e.g., over the course of weeks or months). Such a nucleicacid molecule can additionally comprise a nucleotide sequence encoding apolypeptide that allows for positive selection of engineered cells, orallows for visualization of the engineered cells. In another morespecific embodiment, the nucleotide sequence encodes a polypeptide thatis, e.g., fluorescent under appropriate visualization conditions, e.g.,luciferase (Luc). In a more specific embodiment, such a nucleic acidmolecule can comprise IL-1Ra-DHFR-IRES-Luc, where IL-1Ra isinterleukin-1 receptor antagonist, IRES is an internal ribosomal entrysite, and DHFR is dihydrofolate reductase.

5.9.2 Immortalized Amnion Derived Adherent Cell Lines

Mammalian amnion derived adherent cells can be conditionallyimmortalized by transfection with any suitable vector containing agrowth-promoting gene, that is, a gene encoding a protein that, underappropriate conditions, promotes growth of the transfected cell, suchthat the production and/or activity of the growth-promoting protein isregulatable by an external factor. In a preferred embodiment thegrowth-promoting gene is an oncogene such as, but not limited to, v-myc,N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1aadenovirus or E7 protein of human papillomavirus. In another embodiment,amnion derived adherent cells can be immortalized using cre-loxrecombination, as exemplified for a human pancreatic β-cell line byNarushima, M., et at (Nature Biotechnology, 2005, 23(10:1274-1282).

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells. in one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom ph_(CMV*-1), a minimal promoter from human cytomegalovirus fused totet operator sequences. tTA is a fusion protein of the repressor (tetR)of the transposon-10-derived tet resistance operon of Escherichia coliand the acidic domain of VP16 of herpes simplex virus. Low, non-toxicconcentrations of tet (e.g., 0.01-1.0 μg/mL) almost completely abolishtransactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo^(R)) is one such marker that maybe employed within the present methods. Cells carrying neo^(R) may beselected by means known to those of ordinary skill in the art, such asthe addition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a placental cell culture prepared as described above may be infectedafter, e.g., five days in vitro by incubation for about 20 hours in onevolume of conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least 30% of the cells to doublein a 24 hour period. Preferably, the substrate is apolyornithine/laminin substrate, consisting of tissue culture plasticcoated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), apolylysine/laminin substrate or a surface treated with fibronectin.Cultures are then fed every 3-4 days with growth medium, which may ormay not be supplemented with one or more proliferation-enhancingfactors. Proliferation-enhancing factors may be added to the growthmedium when cultures are less than 50% confluent.

The conditionally-immortalized amnion derived adherent cell lines can bepassaged using standard techniques, such as by trypsinization, when80-95% confluent. Up to approximately the twentieth passage, it is, insome embodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedadherent cell line prepared as described above. In general, such clonalcell lines may be isolated using standard techniques, such as by limitdilution or using cloning rings, and expanded. Clonal cell lines maygenerally be fed and passaged as described above.

Conditionally-immortalized human amnion derived adherent cells lines,which may, but need not, be clonal, may generally be induced todifferentiate by suppressing the production and/or activity of thegrowth-promoting protein under culture conditions that facilitatedifferentiation. For example, if the gene encoding the growth-promotingprotein is under the control of an externally-regulatable promoter, theconditions, e.g., temperature or composition of medium, may be modifiedto suppress transcription of the growth-promoting gene. For thetetracycline-controlled gene expression system discussed above,differentiation can be achieved by the addition of tetracycline tosuppress transcription of the growth-promoting gene. In general, 1 μg/mLtetracycline for 4-5 days is sufficient to initiate differentiation. Topromote further differentiation, additional agents may be included inthe growth medium.

5.10 Methods of Treatment Using Amnion Derived Adherent Cells

5.10.1 Circulatory System Diseases

The amnion derived adherent cells, populations of such cells, andpopulations of cells comprising amnion derived adherent cells, providedherein, can be used to treat individuals exhibiting a variety of diseasestates or conditions that would benefit from angiogenesis. Examples ofsuch disease states or conditions include myocardial infarction, stroke,congestive heart failure, peripheral artery disease, hypoplastic leftheart syndrome, diabetic ulcer, decubitus ulcer, venous ulcer, arterialulcer, burn, non-union fracture, tumor-associated bone loss,osteoarthritis and maxillofacial bone repair. The amnion derivedadherent cells, and populations of such cells, can also be used topromote angiogenesis in individuals exhibiting traumatic tissue loss orto prevent scar formation, or in individuals having total jointreplacement or dental prosthetics.

In a more specific embodiment, the amnion derived adherent cells, andpopulations of such cells, provided herein, can be used to treat anindividual having an insufficiency of the circulatory system, e.g., andindividual having peripheral vascular disease or coronary arterydisease.

In one aspect, provided herein are methods for treating a patient with aheart disease or injury comprising administering a therapeutic cellcomposition to a patient with a disease or injury of the heart orcirculatory system, and evaluating the patient for improvements incardiac function, wherein said cell composition comprises amnion derivedadherent cells as described herein. In one embodiment, the heart diseaseis a cardiomyopathy. In specific embodiments, the cardiomyopathy iseither idiopathic or a cardiomyopathy with a known cause. In otherspecific embodiments, the cardiomyopathy is either ischemic ornonischemic in nature. In another embodiments, the disease of the heartor circulatory system comprises one or more of angioplasty, aneurysm,angina (angina pectoris), aortic stenosis, aortitis, arrhythmias,arteriosclerosis, arteritis, asymmetric septal hypertrophy (ASH),atherosclerosis, atrial fibrillation and flutter, bacterialendocarditis, Barlow's Syndrome (mitral valve prolapse), bradycardia,Buerger's Disease (thromboangiitis obliterans), cardiomegaly,cardiomyopathy, carditis, carotid artery disease, coarctation of theaorta, congenital heart diseases (congenital heart defects), congestiveheart failure (heart failure), coronary artery disease, Eisenmenger'sSyndrome, embolism, endocarditis, erythromelalgia, fibrillation,fibromuscular dysplasia, heart block, heart murmur, hypertension,hypotension, idiopathic infantile arterial calcification, KawasakiDisease (mucocutaneous lymph node syndrome, mucocutaneous lymph nodedisease, infantile polyarteritis), metabolic syndrome, microvascularangina, myocardial infarction (heart attacks), myocarditis, paroxysmalatrial tachycardia (PAT), periarteritis nodosa (polyarteritis,polyarteritis nodosa), pericarditis, peripheral vascular disease,critical limb ischemia, diabetic vasculopathy, phlebitis, pulmonaryvalve stenosis (pulmonic stenosis), Raynaud's Disease, renal arterystenosis, renovascular hypertension, rheumatic heart disease, septaldefects, silent ischemia, syndrome X, tachycardia, Takayasu's Arteritis,Tetralogy of Fallot, transposition of the great vessels, tricuspidatresia, truncus arteriosus, valvular heart disease, varicose ulcers,varicose veins, vasculitis, ventricular septal defect,Wolff-Parkinson-White Syndrome, or endocardial cushion defect.

In other embodiments, the disease of the heart or circulatory systemcomprises one or more of acute rheumatic fever, acute rheumaticpericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis,chronic rheumatic heart diseases, diseases of the mitral valve, mitralstenosis, rheumatic mitral insufficiency, diseases of aortic valve,diseases of other endocardial structures, ischemic heart disease (acuteand subacute), angina pectoris, diseases of pulmonary circulation (acutepulmonary heart disease, pulmonary embolism, chronic pulmonary heartdisease), kyphoscoliotic heart disease, myocarditis, endocarditis,endomyocardial fibrosis, endocardial fibroelastosis, atrioventricularblock, cardiac dysrhythmias, myocardial degeneration, diseases of thecirculatory system including cerebrovascular disease, occlusion andstenosis of precerebral arteries, occlusion of cerebral arteries,diseases of arteries, arterioles and capillaries (atherosclerosis,aneurysm), or diseases of veins and lymphatic vessels.

In one embodiment, treatment comprises treatment of a patient with acardiomyopathy with a therapeutic cell composition comprising amnionderived adherent cells, either with or without another cell type. Inother preferred embodiments, the patient experiences benefits from thetherapy, for example from the ability of the cells to support the growthof other cells, including stem cells or progenitor cells present in theheart, from the tissue ingrowth or vascularization of the tissue, andfrom the presence of beneficial cellular factors, chemokines, cytokinesand the like, but the cells do not integrate or multiply in the patient.In another embodiment, the patient benefits from the therapeutictreatment with the cells, but the cells do not survive for a prolongedperiod in the patient. In one embodiment, the cells gradually decline innumber, viability or biochemical activity, in other embodiments, thedecline in cells may be preceded by a period of activity, for examplegrowth, division, or biochemical activity. In other embodiments,senescent, nonviable or even dead cells are able to have a beneficialtherapeutic effect.

Improvement in an individual having a disease or disorder of thecirculatory system, wherein the individual is administered the amnionderived adherent cells or therapeutic compositions provided herein, canbe assessed or demonstrated by detectable improvement in one or moresymptoms of the disease or disorder of the circulatory system.

In another embodiment, improvement in an individual having a disease ordisorder of the circulatory system, wherein the individual isadministered the amnion derived adherent cells or therapeuticcompositions provided herein, can be assessed or demonstrated bydetectable improvement in one or more, indicia of cardiac function, forexample, demonstration of detectable improvement in one or more of chestcardiac output (CO), cardiac index (CI), pulmonary artery wedgepressures (PAWP), and cardiac index (CI), % fractional shortening (%FS), ejection fraction (EF), left ventricular ejection fraction (LVEF);left ventricular end diastolic diameter (LVEDD), left ventricular endsystolic diameter (LVESD), contractility (e.g. dP/dt), pressure-volumeloops, measurements of cardiac work, an increase in atrial orventricular functioning; an increase in pumping efficiency, a decreasein the rate of loss of pumping efficiency, a decrease in loss ofhemodynamic functioning; and a decrease in complications associated withcardiomyopathy, as compared to the individual prior to administration ofamnion derived adherent cells.

Improvement in an individual receiving the therapeutic compositionsprovided herein can also be assessed by subjective metrics, e.g., theindividual's self-assessment about his or her state of health followingadministration.

Success of administration of the cells is not, in certain embodiments,based on survival in the individual of the administered amnion derivedadherent cells. Success is, instead, based on one or more metrics ofimprovement in cardiac or circulatory health, as noted above. Thus, thecells need not integrate and beat with the patient's heart, or intoblood vessels.

In certain embodiments, the methods of treatment provided hereincomprise inducing the therapeutic amnion derived adherent cells todifferentiate along mesenchymal lineage, e.g., towards a cardiomyogenic,angiogenic or vasculogenic phenotype, or into cells such as myocytes,cardiomyocytes, endothelial cells, myocardial cells, epicardial cells,vascular endothelial cells, smooth muscle cells (e.g. vascular smoothmuscle cells).

Administration of amnion derived adherent cells, or therapeuticcompositions comprising such cells, to an individual in need thereof,can be accomplished, e.g., by transplantation, implantation (e.g., ofthe cells themselves or the cells as part of a matrix-cell combination),injection (e.g., directly to the site of the disease or condition, forexample, directly to an ischemic site in the heart of an individual whohas had a myocardial infarction), infusion, delivery via catheter, orany other means known in the art for providing cell therapy.

In one embodiment, the therapeutic cell compositions are provided to anindividual in need thereof, for example, by injection into one or moresites in the individual. In a specific embodiment, the therapeutic cellcompositions are provided by intracardiac injection, e.g., to anischemic area in the heart. In other specific embodiments, the cells areinjected onto the surface of the heart, into an adjacent area, or evento a more remote area. In preferred embodiments, the cells can home tothe diseased or injured area.

An individual having a disease or condition of the coronary or vascularsystems can be administered amnion derived adherent cells at any timethe cells would be therapeutically beneficial. In certain embodiments,for example, the cells or therapeutic compositions of the invention areadministered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 days of the myocardial infarction. Administrationproximal in time to a myocardial infarction, e.g., within 1-3 or 1-7days, is preferable to administration distal in time, e.g., after 3 or 7days after a myocardial infarction. In other embodiments, the cells ortherapeutic compositions of the invention are administered within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days ofinitial diagnosis of the disease or condition.

Also provided herein are kits for use in the treatment of myocardialinfarction. The kits provide the therapeutic cell composition which canbe prepared in a pharmaceutically acceptable form, for example by mixingwith a pharmaceutically acceptable carrier, and an applicator, alongwith instructions for use. Ideally the kit can be used in the field, forexample in a physician's office, or by an emergency care provider to beapplied to a patient diagnosed as having had a myocardial infarction orsimilar cardiac event.

In specific embodiments of the methods of treatment provided herein, theamnion derived adherent cells are administered with stem cells (that is,stem cells that are not amnion derived adherent cells), myoblasts,myocytes, cardiomyoblasts, cardiomyocytes, or progenitors of myoblasts,myocytes, cardiomyoblasts, and/or cardiomyocytes.

In a specific embodiment, the methods of treatment provided hereincomprise administering amnion derived adherent cells, e.g., atherapeutic composition comprising the cells, to a patient with adisease of the heart or circulatory system; and evaluating the patientfor improvements in cardiac function, wherein the therapeutic cellcomposition is administered as a matrix-cell complex. In certainembodiments, the matrix is a scaffold, preferably bioabsorbable,comprising at least the cells.

To this end, provided herein are populations of amnion derived adherentcells incubated in the presence of one or more factors which stimulatestem or progenitor cell differentiation along a cardiogenic, angiogenic,hemangiogenic, or vasculogenic pathway. Such factors are known in theart; determination of suitable conditions for differentiation can beaccomplished with routine experimentation. Such factors include, but arenot limited to factors, such as growth factors, chemokines, cytokines,cellular products, demethylating agents, and other stimuli which are nowknown or later determined to stimulate differentiation, for example ofstem cells, along cardiogenic, angiogenic, hemangiogenic, orvasculogenic pathways or lineages.

Amnion derived adherent cells may be differentiated along cardiogenic,angiogenic, hemangiogenic, or vasculogenic pathways or lineages byculture of the cells in the presence of factors comprising at least oneof a demethylation agent, a BMP, FGF, Wnt factor protein, Hedgehog,and/or anti-Wnt factors.

Inclusion of demethylation agents tends to allow the cells todifferentiate along mesenchymal lines, toward a cardiomyogenic pathway.Differentiation can be determined by, for example, expression of atleast one of cardiomyosin, skeletal myosin, or GATA4; or by theacquisition of a beating rhythm, spontaneous or otherwise induced; or bythe ability to integrate at least partially into a patient's cardiacmuscle without inducing arrhythmias. Demethylation agents that can beused to initiate such differentiation include, but are not limited to,5-azacytidine, 5-aza-2′-deoxycytidine, dimethylsulfoxide, chelerythrinechloride, retinoic acid or salts thereof, 2-amino-4-(ethylthio)butyricacid, procainamide, and procaine.

In certain embodiments herein, cells induced with one or more factors asidentified above may become cardiomyogenic, angiogenic, hemangiogenic,or vasculogenic cells, or progenitors. Preferably at least some of thecells can integrate at least partially into a recipient's cardiovascularsystem, including but not limited to heart muscle, vascular and otherstructures of the heart, cardiac or peripheral blood vessels, and thelike. In certain other embodiments, the differentiated amnion derivedadherent cells differentiate into cells acquiring two or more of theindicia of cardiomyogenic cells or their progenitors, and able topartially or fully integrate into a recipient's heart or vasculature. Inspecific embodiments, the cells, which administered to an individual,result in no increase in arrhythmias, heart defects, blood vesseldefects or other anomalies of the individual's circulatory system orhealth. In certain embodiments, the amnion derived adherent cells act topromote the differentiation of stem cells naturally present in thepatient's cardiac muscle, blood vessels, blood and the like tothemselves differentiate into for example, cardiomyocytes, or at leastalong cardiomyogenic, angiogenic, hemangiogenic, or vasculogenic lines.

Amnion derived adherent cells, and populations of such cells, can beprovided therapeutically or prophylactically to an individual, e.g., anindividual having a disease, disorder or condition of, or affecting, theheart or circulatory system. Such diseases, disorders or conditions caninclude congestive heart failure due to atherosclerosis, cardiomyopathy,or cardiac injury, e.g., an ischemic injury, such as from myocardialinfarction or wound (acute or chronic).

In certain embodiments, the individual is administered a therapeuticallyeffective amount of amnion derived adherent cells, e.g., in a populationof cells that comprise the amnion derived adherent cells. In a specificembodiment, the population comprises about 50% amnion derived adherentcells. In another specific embodiment, the population is a substantiallyhomogeneous population of amnion derived adherent cells. In otherembodiments the population comprises at least about 5%, 10%, 20%, 25%,30%, 33%, 40%, 60%, 66%, 70%, 75%, 80%, or 90% amnion derived adherentcells.

The amnion derived adherent cells may be administered to an individualin the form of a therapeutic composition comprising the cells andanother therapeutic agent, such as insulin-like growth factor (IGF),platelet-derived growth factor (PDGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF), IL-8, an antithrombogenic agent(e.g., heparin, heparin derivatives, urokinase, and PPack(dextrophenylalanine proline arginine chloromethylketone); antithrombincompounds, platelet receptor antagonists, anti-thrombin antibodies,anti-platelet receptor antibodies, aspirin, dipyridamole, protamine,hirudin, prostaglandin inhibitors, and/or platelet inhibitors), anantiapoptotic agent (e.g., EPO, EPO derivatives and analogs, and theirsalts, TPO, IGF-I, IGF-II, hepatocyte growth factor (HGF), or caspaseinhibitors), an anti-inflammatory agent (e.g., P38 MAP kinaseinhibitors, statins, IL-6 and IL-1 inhibitors, Pemirolast, Tranilast,Remicade, Sirolimus, nonsteroidal anti-inflammatory compounds, forexample, acetylsalicylic acid, ibuprofen, Tepoxalin, Tolmetin, orSuprofen), an immunosuppressive or immunomodulatory agent (e.g.,calcineurin inhibitors, for example cyclosporine, Tacrolimus, mTORinhibitors such as Sirolimus or Everolimus; anti-proliferatives such asazathioprine and mycophenolate mofetil; corticosteroids, e.g.,prednisolone or hydrocortisone; antibodies such as monoclonalanti-IL-2Ra receptor antibodies, Basiliximab, Daclizuma, polyclonalanti-T-cell antibodies such as anti-thymocyte globulin (ATG),anti-lymphocyte globulin (ALG), and the monoclonal anti-T cell antibodyOKT3, or adherent placental stem cells as described in U.S. Pat. No.7,468,276, and U.S. patent application Publication No. and 2007/0275362,the disclosures of which are incorporated herein by reference in theirentireties), and/or an antioxidant (e.g., probucol; vitamins A, C, andE, coenzyme Q-10, glutathione, L cysteine, N-acetylcysteine, orantioxidant derivative, analogs or salts of the foregoing). In certainembodiments, therapeutic compositions comprising the amnion derivedadherent cells further comprise one or more additional cell types, e.g.,adult cells (for example, fibroblasts or endodermal cells), or stem orprogenitor cells. Such therapeutic agents and/or one or more additionalcells, can be administered to an individual in need thereof individuallyor in combinations or two or more such compounds or agents.

In certain embodiments, the individual to be treated is a mammal. In aspecific embodiment the individual to be treated is a human. In specificembodiments, the individual is a livestock animal or a domestic animal.In other specific embodiments, the individual to be treated is a horse,sheep, cow or steer, pig, dog or cat.

5.10.2 Stroke and Other Ischemic Disease

In certain embodiments, provided herein is a method of treating anindividual having a disruption of blood flow, e.g., in or around thebrain, or in the peripheral vasculature, comprising administering to theindividual a therapeutically-effective amount of AMDACs. In certainspecific embodiments, the ischemia is peripheral arterial disease (PAD),e.g., is critical limb ischemia (CLI). In certain other embodiments, theischemia is an ischemia of the central nervous system (CNS). In certainother embodiments, the ischemia is peripheral arterial disease, ischemicvascular disease, ischemic heart disease, ischemic brain disease, orischemic renal disease.

In a specific embodiment, said disruption of flow of blood is a stroke.In a more specific embodiment, said stroke is an ischemic stroke. Inanother more specific embodiment, said stroke is a hemorrhagic stroke,e.g., an intracranial cerebral hemorrhage or spontaneous subarachnoidhemorrhage. In another specific embodiment, said disruption is ahematoma. In more specific embodiments, the hematoma is a duralhematoma, a subdural hematoma or a subarachnoid hematoma. In anotherspecific embodiment, said hematoma is caused by external force on theskull, e.g., a head injury. In another specific embodiment, saiddisruption is a Transient Ischemic Attack (TIA), e.g., recurrent TIA. Inanother specific embodiment, said disruption is a vasospasm, e.g., avasospasm following a hemorrhagic stroke.

In another specific embodiment of the method, said therapeuticallyeffective amount is a number of AMDACs that results in elimination of, adetectable improvement in, lessening of the severity of, or slowing ofthe progression of one or more symptoms of, or neurological deficitsattributable to, a disruption of the flow of blood in or around thebrain or CNS exhibited by said individual, e.g., anoxic injury orhypoxic injury. In another specific embodiment, said therapeuticallyeffective amount of isolated AMDACs is administered to said individualprophylactically, e.g., to reduce or eliminate neurological damagecaused by a second or subsequent disruption of flow of blood in oraround the brain or CNS following said disruption of flow of blood.

In another specific embodiment, said symptom of disruption of blood flowin or around the brain, e.g., stroke, anoxic injury or hypoxic injury,is one or more of hemiplegia (paralysis of one side of the body);hemiparesis (weakness on one side of the body); muscle weakness of theface; numbness; reduction in sensation; altered sense of smell, sense oftaste, hearing, or vision; loss of smell, taste, hearing, or vision;drooping of an eyelid (ptosis); detectable weakness of an ocular muscle;decreased gag reflex; decreased ability to swallow; decreased pupilreactivity to light; decreased sensation of the face; decreased balance;nystagmus; altered breathing rate; altered heart rate; weakness insternocleidomastoid muscle with decreased ability or inability to turnthe head to one side; weakness in the tongue; aphasia (inability tospeak or understand language); apraxia (altered voluntary movements); avisual field defect; a memory deficit; hemineglect or hemispatialneglect (deficit in attention to the space on the side of the visualfield opposite the lesion); disorganized thinking; confusion;development of hypersexual gestures; anosognosia (persistent denial ofthe existence of a deficit); difficulty walking; altered movementcoordination; vertigo; disequilibrium; loss of consciousness; headache;and/or vomiting.

In another specific embodiment, the methods of treatment described abovecomprise administering a second therapeutic agent to said individual. Ina more specific embodiment, said second therapeutic agent is aneuroprotective agent. In a more specific embodiment, said secondtherapeutic agent is NXY-059 (a disulfonyl derivative ofphenylbutylnitrone: disodium4-((tert-butylimino)-methyl)benzene-1,3-disulfonate N-oxide, or disodium4-((oxido-tert-butyl-azaniumylidene)methyl)benzene-1,3-disulfonate; alsoknown as disufenton). In another more specific embodiment, the secondtherapeutic agent is a thrombolytic agent. In a more specificembodiment, said thrombolytic agent is tissue plasminogen activator(tPA). In embodiments in which the disruption of flow of blood in oraround the brain is a hemorrhage, the second therapeutic agent can be anantihypertensive drug, e.g., a beta blocker or diuretic drug, acombination of a diuretic drug and a potassium-sparing diuretic drug, acombination of a beta blocker and a diuretic drug, a combination of anangiotensin-converting enzyme (ACE) inhibitor and a diuretic, anangiotensin-II antagonist and a diuretic drug, and/or a calcium channelblocker and an ACE inhibitor. In another more specific embodiment, thesecond therapeutic agent is a calcium channel blocker, glutamateantagonist, gamma aminobutyric acid (GABA) agonist, an antioxidant orfree radical scavenger.

In another specific embodiment of the method of treatment, said isolatedAMDACs are administered to said individual within 21-30, e.g., 21 daysof development of one or more symptoms of a disruption of the flow ofblood in or around the brain of said individual, e.g., within 21-30,e.g., 21 days of development of symptoms of stroke, anoxic injury orhypoxic injury. In another specific embodiment of the method oftreatment, said isolated AMDACs are administered to said individualwithin 14 days of development of one or more symptoms of a disruption ofthe flow of blood in or around the brain of said individual. In anotherspecific embodiment of the method of treatment, said isolated AMDACs areadministered to said individual within 7 days of development of one ormore symptoms of a disruption of the flow of blood in or around thebrain of said individual. In another specific embodiment of the methodof treatment, said isolated AMDACs are administered to said individualwithin 48 hours of development of one or more symptoms of a disruptionof the flow of blood in or around the brain of said individual. Inanother specific embodiment, said isolated AMDACs are administered tosaid individual within 24 hours of development of one or more symptomsof a disruption of the flow of blood in or around the brain of saidindividual. In another specific embodiment, said isolated AMDACs areadministered to said individual within 12 hours of development of one ormore symptoms of a disruption of the flow of blood in or around thebrain of said individual. In another specific embodiment, said isolatedAMDACs are administered to said individual within 3 hours of developmentof one or more symptoms of a disruption of the flow of blood in oraround the brain of said individual.

In a specific embodiment, said disruption of flow of blood is criticallimb ischemia. In another more specific embodiment, said CLI is a severeblockage in the arteries of the lower extremities, which markedlyreduces blood-flow. In another more specific embodiment said CLI ischaracterized by ischemic rest pain, severe pain in the legs and feetwhile a person is not moving, non-healing sores on the feet or legs,pain or numbness in the feet, shiny, smooth, dry skin of the legs orfeet, thickening of the toenails, absent or diminished pulse in the legsor feet, open sores, skin infections or ulcers that will not heal, drygangrene (dry, black skin) of the legs or feet. In another specificembodiment, CLI can lead to loss of digits and or whole limbs. Inanother specific embodiment of the method, said therapeuticallyeffective amount is a number of AMDACs that results in elimination of, adetectable improvement in, lessening of the severity of, or slowing ofthe progression of one or more symptoms of, loss of limb function and oroxygen deprivation (hypoxia/anoxia) attributable to, a disruption of theflow of blood in or around the brain or CNS exhibited by saidindividual, e.g., anoxic injury or hypoxic injury. In another specificembodiment, said therapeutically effective amount of isolated AMDACs isadministered to said individual prophylactically, e.g., to reduce oreliminate tissue damage caused by a second or subsequent disruption offlow of blood in or around the limb following said disruption of flow ofblood.

5.10.3 Dosages and Routes of Administration

Administration of AMDACs to an individual in need thereof can be by anymedically-acceptable route relevant for the disease or condition to betreated. In another specific embodiment of the methods of treatmentdescribed above, said AMDACs are administered by bolus injection. Inanother specific embodiment, said isolated AMDACs are administered byintravenous infusion. In a specific embodiment, said intravenousinfusion is intravenous infusion over about 1 to about 8 hours. Inanother specific embodiment, said isolated AMDACs are administeredintracranially. In another specific embodiment, said isolated AMDACs areadministered intramulscularly. In another specific embodiment, saidisolated AMDACs are administered intraperitoneally. In another specificembodiment, said isolated AMDACs are administered intra-arterially. In amore specific embodiment, said isolated AMDACs are administered withinan area of ischemia. In another more specific embodiment, said isolatedAMDACs are administered to an area peripheral to an ischemia. In anotherspecific embodiment of the method of treatment, said isolated AMDACs areadministered intramuscularly, intradermally, or subcutaneously.

In another specific embodiment of the methods of treatment describedabove, said AMDACs are administered once to said individual. In anotherspecific embodiment, said isolated AMDACs are administered to saidindividual in two or more separate administrations. In another specificembodiment, said administering comprises administering between about1×10⁴ and 1×10⁵ isolated AMDACs, e.g., AMDACs per kilogram of saidindividual. In another specific embodiment, said administering comprisesadministering between about 1×10⁵ and 1×10⁶ isolated AMDACs per kilogramof said individual. In another specific embodiment, said administeringcomprises administering between about 1×10⁶ and 1×10⁷ isolated AMDACsper kilogram of said individual. In another specific embodiment, saidadministering comprises administering between about 1×10⁷ and 1×10⁸isolated placental cells per kilogram of said individual. In otherspecific embodiments, said administering comprises administering betweenabout 1×10⁶ and about 2×10⁶ isolated placental cells per kilogram ofsaid individual; between about 2×10⁶ and about 3×10⁶ isolated placentalcells per kilogram of said individual; between about 3×10⁶ and about4×10⁶ isolated placental cells per kilogram of said individual; betweenabout 4×10⁶ and about 5×10⁶ isolated placental cells per kilogram ofsaid individual; between about 5×10⁶ and about 6×10⁶ isolated placentalcells per kilogram of said individual; between about 6×10⁶ and about7×10⁶ isolated placental cells per kilogram of said individual; betweenabout 7×10⁶ and about 8×10⁶ isolated placental cells per kilogram ofsaid individual; between about 8×10⁶ and about 9×10⁶ isolated placentalcells per kilogram of said individual; or between about 9×10⁶ and about1×10⁷ isolated placental cells per kilogram of said individual. Inanother specific embodiment, said administering comprises administeringbetween about 1×10⁷ and about 2×10⁷ isolated placental cells perkilogram of said individual to said individual. In another specificembodiment, said administering comprises administering between about1.3×10⁷ and about 1.5×10⁷ isolated placental cells per kilogram of saidindividual to said individual. In another specific embodiment, saidadministering comprises administering up to about 3×10⁷ isolatedplacental cells per kilogram of said individual to said individual. In aspecific embodiment, said administering comprises administering betweenabout 5×10⁶ and about 2×10⁷ isolated placental cells to said individual.In another specific embodiment, said administering comprisesadministering about 150×10⁶ isolated placental cells in about 20milliliters of solution to said individual.

In a specific embodiment, said administering comprises administeringbetween about 5×10⁶ and about 2×10⁷ isolated placental cells to saidindividual, wherein said cells are contained in a solution comprising10% dextran, e.g., dextran-40, 5% human serum albumin, and optionally animmunosuppressant. In another specific embodiment, said administeringcomprises administering between about 5×10⁷ and 3×10⁹ isolated placentalcells intravenously. In more specific embodiments, said administeringcomprises administering about 9×10⁸ isolated placental cells or about1.8×10⁹ isolated placental cells intravenously. In another specificembodiment, said administering comprises administering between about5×10⁷ and 1×10⁸ isolated placental cells intracranially. In a morespecific embodiment, said administering comprises administering about9×10⁷ isolated placental cells intracranially.

5.11 Differentiation of Amnion Derived Adherent Cells

The amnion derived adherent cells provided herein can be differentiated.In one embodiment, the cell has been differentiated sufficiently forsaid cell to exhibit at least one characteristic of an endothelial cell,a myogenic cell, or a pericytic cell, e.g., by contacting the cell withvascular endothelial growth factor (VEGF), or as described in Sections5.11.2, 6.3.3, or 6.3.4, below. In more specific embodiments, saidcharacteristic of an endothelial cell, myogenic cell or pericytic cellis expression of one or more of CD9, CD31, CD54, CD102, NG2(neural/glial antigen 2) or alpha smooth muscle actin, which isincreased compared to an amniotic cell that is OCT-4⁻, VEGFR2/KDR⁺,CD9⁺, CD54⁺, CD105⁺, CD200⁺, and VE-cadherin⁻. In other more specificembodiments, said characteristic of an endothelial cell, myogenic cellor pericytic cell is expression of one or more of CD9, CD31, CD54,CD102, NG2 (neural/glial antigen 2) or alpha smooth muscle actin, whichis increased compared to an amniotic cell that is OCT-4⁻, VEGFR2/KDR⁺,and VEGFR1/Flt-1⁺.

5.11.1 Induction of Angiogenesis

Angiogenesis from the amnion derived adherent cells provided herein canbe accomplished as follows. The amnion derived adherent cells, arecultured, e.g., in an endothelial cell medium, e.g., EGM®-2 (Lonza) or amedium comprising 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma); 2% fetalcalf serum (Hyclone Labs.); 1× insulin-transferrin-selenium (ITS); 1×linoleic acid-bovine serum albumin (LA-BSA); 5×10⁻⁹M dexamethasone(Sigma); 10⁻⁴ M ascorbic acid 2-phosphate (Sigma); epidermal growthfactor 10 ng/mL (R&D Systems); and platelet-derived growth factor(PDGF-BB) 10 ng/mL (R&D Systems), to passage 3. The cells are thenplated onto MATRIGEL™ or a substrate comprising collagen-1, e.g., in96-well plates at a density of, e.g., about 1.5×10⁴ cells per well inthe same medium or DMEM with FBS (0-5% v/v) comprising vascularendothelial growth factor (VEGF) at, e.g., about 10 to 50 ng permilliliter. Medium can be changed about twice a week. Angiogenesis isevidenced by visual inspection of the cells for sprouting of vessel-likestructures and tube formation, visible under a microscope at amagnification of, e.g., 50× to 100×.

5.11.2 Induction of Differentiation into Cardiac Cells

Myogenic (cardiogenic) differentiation of the amnion derived adherentcells provided herein can be accomplished, for example, by placing thecells in cell culture conditions that induce differentiation intocardiomyocytes. A preferred cardiomyocytic medium comprises DMEM/20% CBSsupplemented with retinoic acid, 1 μL; basic fibroblast growth factor,10 ng/mL; and transforming growth factor beta-1, 2 ng/mL; and epidermalgrowth factor, 100 ng/mL. KnockOut Serum Replacement (Invitrogen,Carlsbad, Calif.) may be used in lieu of CBS. Alternatively, the amnionderived adherent cells are cultured in DMEM/20% CBS supplemented with 1to 100, e.g., 50 ng/mL Cardiotropin-1 for 24 hours. In anotherembodiment, amnion derived adherent cells can be cultured 10-14 days inprotein-free medium for 5-7 days, then stimulated with human myocardiumextract, e.g., produced by homogenizing human myocardium in 1% HEPESbuffer supplemented with 1% cord blood serum.

Differentiation can be confirmed by demonstration of cardiac actin geneexpression, e.g., by RT/PCR, or by visible beating of the cell. Anadherent cell is considered to have differentiated into a cardiac cellwhen the cell displays one or more of these characteristics.

6. EXAMPLES 6.1 Example 1 Isolation and Expansion of Adherent Cells fromAmniotic Membrane

This Example demonstrates the isolation and expansion of amnion derivedadherent cells.

6.1.1 Isolation

Amnion derived adherent cells were isolated from amniotic membrane asfollows. Amnion/chorion were cut from the placenta, and amnion wasmanually separated from the chorion. The amnion was rinsed with sterilePBS to remove residual blood, blood clots and other material. Sterilegauze was used to remove additional blood, blood clots or other materialthat was not removed by rinsing, and the amnion was rinsed again withPBS. Excess PBS was removed from the membrane, and the amnion was cutwith a scalpel into 2″ by 2″ segments. For epithelial cell release, aprocessing vessel was set up by connecting a sterile jacketed glassprocessing vessel to a circulating 37° C. water bath using tubing andconnectors, and set on a stir plate. Trypsin (0.25%, 300 mL) was warmedto 37° C. in the processing vessel; the amnion segments were added, andthe amnion/trypsin suspension was agitated, e.g., at 100 RPM-150 RPM at37° C. for 15 minutes. A sterile screening system was assembled byplacing a sterile receptacle on a sterile field next to the processingvessel and inserting a sterile 75 μm to 125 μm screen into thereceptacle (Millipore, Billerica, Mass.). After agitating the amnionsegments for 15 minutes, the contents of the processing vessel weretransferred to the screen, and the amnion segments were transferred,e.g., using sterile tweezers back into the processing vessel; thetrypsin solution containing the epithelial cells was discarded. Theamnion segments were agitated again with 300 mL trypsin solution (0.25%)as described above. The screen was rinsed with approximately 100-150 mLof PBS, and the PBS solution was discarded. After agitating the amnionsegments for 15 minutes, the contents of the processing vessel weretransferred to the screen. The amnion segments were then transferredback into the processing vessel; the trypsin solution containing theepithelial cells was discarded. The amnion segments were agitated againwith 300 mL trypsin solution (0.25%) as described above. The screen wasrinsed with approximately 100-150 mL of PBS, and the PBS solution wasdiscarded. After agitating the amnion segments for 15 minutes, thecontents of the processing vessel were transferred to the screen. Theamnion segments were then transferred back into the processing vessel,and the trypsin solution containing the epithelial cells was discarded.The amnion segments were agitated in PBS/5% FBS (1:1 ratio of amnion toPBS/5% FBS solution by volume) at 37° C. for approximately 2-5 minutesto neutralize the trypsin. A fresh sterile screen system was assembled.After neutralizing the trypsin, the contents of the processing vesselwere transferred to the new screen, and the amnion segments weretransferred back into the processing vessel. Room temperature, sterilePBS (400 mL) was added to the processing vessel, and the contents of theprocessing vessel were agitated for approximately 2-5 minutes. Thescreen was rinsed with approximately 100-150 mL of PBS. After agitation,the contents of the processing vessel were transferred to the screen;the processing flask was rinsed with PBS, and the PBS solution wasdiscarded. The processing vessel was then filled with 300 mL ofpre-warmed DMEM, and the amnion segments were transferred into the DMEMsolution.

For release of the amnion derived adherent cells, the treated amnioticmembrane was further treated with collagenase as follows. A sterilecollagenase stock solution (500 U/mL) was prepared by dissolving theappropriate amount of collagenase powder (varied with the activity ofthe collagenase lot received from the supplier) in DMEM. The solutionwas filtered through a 0.22 μm filter and dispensed into individualsterile containers. CaCl₂ solution (0.5 mL, 600 mM) was added to each100 mL dose, and the doses were frozen. Collagenase (100 mL) was addedto the amnion segments in the processing vessel, and the processingvessel was agitated for 30-50 minutes, or until amnion digestion wascomplete by visual inspection. After amnion digestion was complete, 100mL of pre-warmed sterile PBS/5% FBS was added to the processing vessel,and the processing vessel was agitated for an additional 2-3 minutes.Following agitation, the contents of the flask were transferred to asterile 60 μm screen, and the liquid was collected by vacuum filtration.The processing vessel was rinsed with 400 mL of PBS, and the PBSsolution was sterile-filtered. The filtered cell suspension was thencentrifuged at 300×g for 15 minutes at 20° C., and the cell pellets wereresuspended in pre-warmed PBS/2% FBS (approximately 10 mL total).

6.1.2 Establishment

Freshly isolated angiogenic amniotic cells were added to growth mediumcontaining 60% DMEM-LG (Gibco); 40% MCBD-201 (Sigma); 2% FBS (HycloneLabs), 1× insulin-transferrin-selenium (ITS); 10 ng/mL linoleicacid-bovine serum albumin (LA-BSA); 1 n-dexamethasone (Sigma); 100 μMascorbic acid 2-phosphate (Sigma); 10 ng/mL epidermal growth factor (R &D Systems); and 10 ng/mL platelet-derived growth factor (PDGF-BB) (R & DSystems) and were plated in a T-Flask at a seeding density of 10,000cells per cm². The culture device(s) were then incubated at 37° C., 5%CO₂ with >90% humidity. Cellular attachment, growth, and morphology weremonitored daily. Non-adherent cells and debris were removed by mediumexchange. Medium exchange was performed twice per week. Adherent cellswith typical fibroblastoid/spindle shape morphology appeared at severaldays after initial plating. When confluency reached 40%-70% (at 4-11days after initial plating), the cells were harvested by trypsinization(0.25% trypsin —EDTA) for 5 minutes at room temperature (37° C.). Afterneutralization with PBS-5% FBS, the cells were centrifuged at 200-400 gfor 5-15 minutes at room temperature, and then were resuspended ingrowth medium. At this point, an AMDAC line was considered to besuccessfully established at the initial passage. Initial passage amnionderived adherent cells were, in some cases, cryopreserved or expanded.

6.1.3 Culture Procedure

Amnion derived adherent cells were cultured in the growth mediumdescribed above and seeded at a density of 2000-4000 per cm² in anappropriate tissue culture-treated culture device(s). The culturedevice(s) were then incubated at 37° C., 5% CO₂ with >90% humidity.During culture, AMDACs would adhere and proliferate. Cellular growth,morphology, and confluency were monitored daily. Medium exchange wasperformed twice a week to replenish fresh nutrients if the cultureextended to 5 days or more. When confluency reached 40%-70% (at 3-7 daysafter seeding), the cells were harvested by trypsinization (0.05%-0.25%trypsin—EDTA) for 5 minutes at room temperature (37° C.). Afterneutralization with PBS-5% FBS, the cells were centrifuged at 200-400 gfor 5-15 minutes at room temperature, then were resuspended in growthmedium.

AMDACs isolated and cultured in this manner typically produced33530+/−15090 colony-forming units (fibroblast) (CFU-F) out of 1×10⁶cells plated.

6.2 Example 2 Phenotypic Characterization of Amnion Derived AdherentCells

6.2.1 Gene and Protein Expression Profiles

This Example describes phenotypic characterization of amnion derivedadherent cells, including characteristic cell surface marker, mRNA, andproteomic expression.

Sample Preparation:

Amnion derived adherent cells were obtained as described in Example 1.The cells at passage 6 were grown to approximately 70% confluence ingrowth medium as described in Example 1, above, trypsinized, and washedin PBS. NTERA-2 cells (American Type Culture Collection, ATCC NumberCRL-1973) were grown in DMEM containing 4.5 g/L glucose, 2 mM glutamineand 10% FBS. Nucleated cell counts were performed to obtain a minimum of2×10⁶ to 1×10⁷ cells. The cells were then lysed using a Qiagen RNeasykit (Qiagen, Valencia, Calif.), utilizing a QIAshredder, to obtain thelysates. The RNA isolation was then performed using a Qiagen RNeasy kit.RNA quantity and quality were determined using a Nanodrop ND 1000spectrophotometer, 25 ng/μL of RNA/reaction. The cDNA reactions wereprepared using an Applied Biosystems (Foster City, Calif.) High CapacitycDNA Archive Kit. Real time PCR reactions were performed using TAQMAN®universal PCR master mixes from Applied Biosystems. Reactions were runin standard mode on an Applied Biosystems 7300 Real time PCR system for40 cycles.

Sample Analysis and Results:

Using the real time PCR methodology and specific TAQMAN® gene expressionprobes and/or the TAQMAN® human angiogenesis array (Applied Biosystems),cells were characterized for expression of stem cell-related, angiogenicand cardiomyogenic markers. Results were expressed either as therelative expression of a gene of interest in comparison to the pertinentcell controls, or the relative expression (delta Ct) of the gene ofinterest in comparison to a ubiquitously expressed housekeeping gene(for example, GAPDH, 18S, or GUSB).

Amnion derived adherent cells expressed various, stem-cell related,angiogenic and cardiomyogenic genes and displayed a relative absence ofOCT-4 expression in comparison to NTERA-2 cells. Table 1 summarizes theexpression of selected angiogenic, cardiomyogenic, and stem cell genes,and FIG. 1 demonstrates the lack of expression in AMDACs of the stemcell-related genes POU5F1 (OCT-4), NANOG, SOX2, NES, DNMT3B, and TERT.

TABLE 1 Gene expression profile of amnion derived adherent cells asdetermined by RT-PCR. AMDAC Marker Positive Negative mRNA ACTA2 X XACTC1 X X ADAMTS1 X X AMOT X X ANG X X ANGPT1 X X ANGPT2 X X ANGPT4 X XANGPTL1 X X ANGPTL2 X X ANGPTL3 X X ANGPTL4 X X BAI1 X X BGLAP X X c-mycX X CD31 X X CD34 X X CD44 X X CD140a X X CD140b X X CD200 X X CD202b XX CD304 X X CD309 (VEGFR2/KDR) X X CDH5 X X CEACAM1 X X CHGA X X COL15A1X X COL18A1 X X COL4A1 X X COL4A2 X X COL4A3 X X Connexin-43 X X CSF3 XX CTGF X X CXCL10 X X CXCL12 X X CXCL2 X X DLX5 X X DNMT3B X X ECGF1 X XEDG1 X X EDIL3 X X ENPP2 X X EPHB2 X X F2 X X FBLN5 X X FGA X X FGF1 X XFGF2 X X FGF4 X X FIGF X X FLT3 X X FLT4 X X FN1 X X FOXC2 X XFollistatin X X Galectin-1 X X GRN X X HEY1 X X HGF X X HLA-G X X HSPG2X X IFNB1 X X IFNG X X IL-8 X X IL-12A X X ITGA4 X X ITGAV X X ITGB3 X XKLF-4 X X LECT1 X X LEP X X MDK X X MMP-13 X X MMP-2 X X MYOZ2 X X NANOGX X NESTIN X X NRP2 X X PDGFB X X PF4 X X PGK1 X X PLG X X POU5F1(OCT-4) X X PRL X X PROK1 X X PROX1 X X PTN X X SEMA3F X X SERPINB5 X XSERPINC1 X X SERPINF1 X X SOX2 X X TERT X X TGFA X X TGFB1 X X THBS1 X XTHBS2 X X TIE1 X X TIMP2 X X TIMP3 X X TNF X X TNFSF15 X X TNMD X XTNNC1 X X TNNT2 X X VASH1 X X VEGF X X VEGFB X X VEGFC X X VEGFR1/FLT-1X X XLKD1 X XColumn “mRNA” indicates that the presence or absence of mRNA forparticular markers were determined in each instance.

In a separate experiment, AMDACs were additionally found to expressgenes for Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2),nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),glial-derived neurotrophic factor (GDNF), neurotrophin 3 (NT-3), NT-5,hypoxia-Inducible Factor 1a (HIF1A), hypoxia-inducible protein 2 (HIG2),heme oxygenase (decycling) 1 (HMOX1), Extracellular superoxide dismutase[Cu—Zn] (SOD3), catalase (CAT), transforming growth factor β1 (TGFB1),transforming growth factor β1 receptor (TGFB1R), and hepatoycte growthfactor receptor (HGFR/c-met).

6.2.2 Flow Cytometry for Evaluation of Angiogenic Potency of AmnionDerived Adherent Cells

Flow cytometry was used as a method to quantify phenotypic markers ofamnion derived adherent cells to define the identity of the cells. Cellsamples were obtained from frozen stocks. Prior to thaw and duringreagent preparation, cell vials were maintained on dry ice.Subsequently, samples were thawed rapidly using a 37° C. water bath.Pre-freeze cell counts were used for calculations for initial post-thawcell number-dependent dilutions. Briefly, cryovials were thawed in a 37°C. water bath for approximately 30 seconds with gentle agitation.Immediately following thawing, approximately 100-200 μL of cold (2 to 8°C.) thawing solution (PBS with 2.5% albumin and 5% Gentran 40) was addedto the cryovial and mixed. After gentle mixing, the total volume in thecryovials was transferred into a 15 mL conical tube containing an equalvolume of cold (2 to 8° C.) thawing solution. The cells were centrifugedin a conical tube at 400 g for 5 minutes at room temperature beforeremoving the supernatant. The residual volume was measured with apipette (estimation); the residual volume and cell pellet wereresuspended at room temperature in 1% FBS in PBS to achieve a cellconcentration of 250×10³ cells/100 μL buffer. For example, 1×10⁶ cellswould be resuspended in 400 μL 1% FBS. The cell suspension was placedinto pre-labeled 5 mL FACS tubes (Becton Dickinson (BD), Franklin Lakes,N.J.). For each primary antibody isotype, 100 μL of cell suspension wasaliquoted into one isotype control tube. Prior to phenotype analysis,the concentrations of all antibodies were optimized to achieve goodsignal to noise ratios and adequate detection of CD antigens across apotential four-log dynamic range. The volume of each isotype and sampleantibody that was used to stain each sample was determined. Tostandardize the amount of antibody (in μg) in the isotype and sampletubes, the concentration of each antibody was calculated as (1/actualantibody concentration (μg/μL))×(desired final quantity of antibody inμg for 2.5×10⁵ cells)=#μL of antibody added. A master mix of antibodiesfor both the isotype and the sample was made with the appropriate amountof antibody added to each tube. The cells were stained for 15-20 minutesat room temperature in the dark. After staining, unbound antibody ineach sample was removed by centrifugation (400 g×5 minutes) followed bywashing using 2 mL 1% FBS PBS (room temperature) before resuspension in150 μL of room temperature 1% FBS PBS. The samples were then analyzed onBecton Dickinson FACSCalibur, FACSCantoI or BD FACSCantoII flowcytometers prepared for use per manufacturer's instructions.Multi-parametric flow cytometry data sets (side scatter (SSC), forwardscatter (FSC) and integrated fluorescence profiles (FL)) were acquiredwithout setting on-the-fly instrument compensation parameters.Compensation parameters were determined after acquisition using theFACSDiva software according to the manufacturer's instructions. Theseinstrument settings were applied to each sample. Fluorophore conjugatesused in these studies were Allophycocyanin (APC), AlexaFluor 647(AF647), Fluorescein isothiocyanate (FITC), Phycoerythrin (PE) andPeridinin chlorophyll protein (PerCP), all from BD Biosciences. Table 2summarizes the expression of selected cell-surface markers, includingangiogenic markers.

TABLE 2 Cell surface marker expression in amnion derived adherent cellsas determined by flow cytometry. Immunolocalization AMDAC MarkerPositive Negative Flow Cytometry CD6 X X CD9 X X CD10 X X CD31 X X CD34X X CD44 X X CD45 X X CD49b X X CD49c X X CD49d X X CD54 X X CD68 X XCD90 X X CD98 X X CD105 X X CD117 X X CD133 X X CD143 X X CD144(VE-cadherin) X X CD146 X X CD166 X X CD184 X X CD200 X X CD202b X XCD271 X X CD304 X X CD309 (VEGFR2/KDR) X X CD318 X X CD349 X X CytoK X XHLA-ABC + X X B2 Micro+ Invariant Chain + X X HLA-DR-DP-DQ+ PDL-1 X XVEGFR1/FLT-1 X X XColumn “Immunolocalization Flow Cytometry” indicates that the presenceor absence of particular markers were determined by immunolocalization,specifically flow cytometry.

In another experiment, AMDAC cells were labeled with anti-human CD49f(Clone GoH3, phycoerythrin-conjugated; BD Pharmingen Part No. 555736),and analyzed by flow cytometry. Approximately 96% of the AMDACs labeledwith anti-CD49f (that is, were CD49f⁺).

In other experiments, AMDACs were additionally found byimmunolocalization to express CD49a, CD106, CD119, CD130, c-met(hepatocyte growth factor receptor; HGFR), CXC chemokine receptor 1(CXCR1), PDGFRA, and PDGFRB by immunolocalization. AMDACs were alsofound, by immunolocalization, to lack expression of CD49e, CD62E,fibroblast growth factor receptor 3 (FGFR3), tumor necrosis factorreceptor superfamily member 12A (TNFRSF12A), insulin-like growth factor1 receptor (IGF-1R), CXCR2, CXCR3, CXCR4, CXCR6, chemokine receptor 1(CCR1), CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, epidermal growthfactor receptor (EGF-R), insulin receptor (CD220), interleukin receptor4 (IL4-R; CD124), IL6-R (CD126), TNF-R1α and 1b (CD120a, b), anderbB2/Her2.

6.2.3 Immunohistochemistry (IHC)/Immunofluorochemistry (IFC) forEvaluation of Angiogenic Potency of Amnion Derived Adherent Cells

Amnion derived adherent cells from passage 6 were grown to approximately70% confluence on 4-well chamber slides and fixed with a 4% formalinsolution for 30 minutes each. After fixation, the slides were rinsedwith PBS two times for 5 minutes. The slides were then incubated with10% normal serum from the same host as the secondary antibody, 2×casein, and 0.3% Triton X100 in PBS, for 20 minutes at room temperaturein a humid chamber. Excess serum was blotted off and the slides wereincubated with primary antibody (goat polyclonal IgG (Santa Cruz; SantaCruz, Calif.) in a humidified chamber. Time and temperature forincubations were determined by selecting the optimal conditions for theantibody being used. In general, incubation times were 1 to 2 hours at37° C. or overnight at 4° C. The slides were then rinsed with PBS threetimes for 5 minutes each and incubated for 20-30 minutes at roomtemperature in a humid chamber with fluorescent-conjugatedanti-immunoglobulin secondary antibody directed against the host of theprimary antibody (rabbit anti-goat antibody (Santa Cruz)). Thereafter,the slides were rinsed with PBS three times for 5 minutes each, mountedwith a coverslip utilizing DAPI VECTASHIELD® (Vector Labs) mountingsolution to counterstain nuclei. Cell staining was visualized utilizinga Nikon fluorescence microscope. All pictures were taken at equalexposure time normalized against the background of the correspondingisotype (goat IgG (Santa Cruz)). Table 3 summarizes the results for theexpression of angiogenic proteins by amnion derived adherent cells.

TABLE 3 Angiogenic markers present or absent on amnion derived adherentcells. Immunolocalization Immunofluorescence AMDAC Marker PositiveNegative Immunohistochemistry CD31 X X CD34 X X (VEGFR2/KDR) X XConnexin-43 X X Galectin-1 X X TEM-7 X X

Amnion derived adherent cells expressed the angiogenic marker tumorendothelial marker 7 (TEM-7), one of the proteins shown in Table 3. SeeFIG. 2.

6.2.4 Membrane Proteomics for Evaluation of Angiogenic Potency of AmnionDerived Adherent Cells

Membrane Protein Purification: Cells at passage 6 were grown toapproximately 70% confluence in growth medium, trypsinized, and washedin PBS. The cells were then incubated for 15 minutes with a solutioncontaining protease inhibitor cocktail (P8340, Sigma Aldrich, St. Louis,Mo.) prior to cell lysis. The cells were then lysed by the addition of a10 mM HCl solution (thus avoiding the use of detergents) and centrifugedfor 10 minutes at 400 g to pellet and remove the nuclei. Thepost-nuclear supernatant was transferred to an ultracentrifugation tubeand centrifuged using a WX80 ultracentrifuge with a T-1270 rotor (ThermoFisher Scientific, Asheville, N.C.) at 100,000 g for 150 minutesgenerating a membrane protein pellet.

Generation, Immobilization and Digestion of Proteoliposomes:

The membrane protein pellet was washed several times using Nanoxisbuffer (10 mM Tris, 300 mM NaCl, pH 8). The membrane protein pellet wassuspended in 1.5 mL of Nanoxis buffer and then tip-sonicated using aVIBRA-CELL™ VC505 ultrasonic processor (Sonics & Materials, Inc.,Newtown, Conn.) for 20 minutes on ice. The size of the proteoliposomeswas determined by staining with FM1-43 dye (Invitrogen, Carlsbad,Calif.) and visualization with fluorescence microscopy. The proteinconcentration of the proteoliposome suspension was determined by a BCAassay (Thermo Scientific). The proteoliposomes were then injected ontoan LPI™Flow Cell (Nanoxis AB, Gothenburg, Sweden) using a standardpipette tip and allowed to immobilize for 1 hour. After immobilization,a series of washing steps were carried out and trypsin at 5 μg/mL(Princeton Separations, Adelphi, N.J.) was injected directly onto theLPI™ Flow Cell. The chip was incubated overnight at 37° C. and thetryptic peptides were eluted from the LPI™ chip and then desalted usinga Sep-Pak cartridge (Waters Corporation, Milford, Mass.).

LTQ Linear Ion Trap LC/MS/MS Analysis:

Each tryptic digest sample was separated on a 0.2 mm×150 mm 3 μm 200 ÅMAGIC C18 column (Michrom Bioresources, Inc., Auburn, Calif.) that wasinterfaced directly to an axial desolvation vacuum-assistednanocapillary electrospray ionization (ADVANCE) source (MichromBioresources, Inc.) using a 180 minute gradient (Buffer A: Water, 0.1%Formic Acid; Buffer B: Acetonitrile, 0.1% Formic Acid). The ADVANCEsource achieves a sensitivity that is comparable to traditional nanoESIwhile operating at a considerably higher flow rate of 3 μL/min. Elutedpeptides were analyzed on an LTQ linear ion trap mass spectrometer(Thermo Fisher Scientific, San Jose, Calif.) that employed tendata-dependent MS/MS scans following each full scan mass spectrum. Sevenanalytical replicate datasets were collected for each biological sample.

Bioinformatics:

Seven RAW files corresponding to the 7 analytical replicate datasetsthat were collected for each cell line were searched as a single searchagainst the IPI Human Database using an implementation of the SEQUESTalgorithm on a Sorcerer Solo™ workstation (Sage-N Research, San Jose,Calif.). A peptide mass tolerance of 1.2 amu was specified, oxidation ofmethionine was specified as a differential modification, andcarbamidomethylation was specified as a static modification. Scaffoldsoftware implementation of the Trans-Proteomic Pipeline (TPP) was usedto sort and parse the membrane proteomic data. Proteins were consideredfor analysis if they were identified with a peptide probability of 95%,protein probability of 95% and 1 unique peptide. Comparisons betweenmembrane proteomic datasets were made using custom Perl scriptsdeveloped in-house.

Results:

As shown in Table 4, amnion derived adherent cells expressed variousangiogenic and cardiomyogenic markers.

TABLE 4 Cardiomyogenic or angiogenic markers expressed by amnion derivedadherent cells. Immunolocalization Membrane AMDAC Marker PositiveNegative Proteomics Activin receptor type IIB X X ADAM 17 X XAlpha-actinin 1 X X Angiotensinogen X X Filamin A X X Macrophageacetylated X X LDL receptor I and II Megalin X X Myosin heavy chain nonX X muscle type A Myosin-binding protein X X C cardiac type Wnt-9 X X

6.2.5 Secretome Profiling for Evaluation of Angiogenic Potency of AmnionDerived Adherent Cells

Protein Arrays:

Amnion derived adherent cells at passage 6 were plated at equal cellnumbers in growth medium and conditioned media were collected after 4days. Simultaneous qualitative analysis of multiple angiogeniccytokines/growth factors in cell-conditioned media was performed usingRayBiotech Angiogenesis Protein Arrays (Norcross, Ga.). In brief,protein arrays were incubated with 2 mL 1× Blocking Buffer (Ray Biotech)at room temperature for 30 minutes (min) to block membranes.Subsequently, the Blocking Buffer was decanted and the membranes wereincubated with 1 mL of sample (growth medium conditioned by therespective cells for 4 days) at room temperature for 1 to 2 hours. Thesamples were then decanted and the membranes were washed 3×5 min with 2mL of 1× Wash Buffer I (Ray Biotech) at room temperature with shaking.Then, the membranes were washed 2×5 min with 2 mL of 1× Wash Buffer II(Ray Biotech) at room temperature with shaking. Thereafter, 1 mL ofdiluted biotin-conjugated antibodies (Ray Biotech) was added to eachmembrane and incubated at room temperature for 1-2 hours and washed withthe Wash Buffers as described above. Diluted HRP-conjugated streptavidin(2 mL) was then added to each membrane and the membranes were incubatedat room temperature for 2 hours. Finally, the membranes were washedagain, incubated with the ECL™ detection kit (Amersham) according tospecifications and the results were visualized and analyzed using theKodak Gel Logic 2200 Imaging System. The secretion of various angiogenicproteins by AMDACs is shown in FIG. 3.

ELISAs:

Quantitative analysis of single angiogenic cytokines/growth factors incell-conditioned media was performed using commercially available kitsfrom R&D Systems (Minneapolis, Minn.). In brief, ELISA assays wereperformed according to manufacturer's instructions and the amount of therespective angiogenic growth factors in the conditioned media wasnormalized to 1×10⁶ cells. Amnion derived adherent cells (n=6) exhibitedapproximately 4500 pg VEGF per million cells and approximately 17,200 pgIL-8 per million cells.

TABLE 5 ELISA results for angiogenic markers Secretome Analysis ELISA,AMDAC Marker Positive Negative Protein Arrays ANG X X EGF X X ENA-78 X XFGF2 X X Follistatin X X G-CSF X X GRO X X HGF X X IL-6 X X IL-8 X XLeptin X X MCP-1 X X MCP-3 X X PDGFB X X PLGF X X Rantes X X TGFB1 X XThrombopoietin X X TIMP1 X X TIMP2 X X uPAR X X VEGF X X VEGFD X X

In a separate experiment, AMDACs were confirmed to also secreteangiopoietin-1, angiopoietin-2, PECAM-1 (CD31; platelet endothelial celladhesion molecule), laminin and fibronectin.

6.2.6 AMDAC MicroRNA Expression Confirms Angiogenic Activity

This Example demonstrates that AMDACs express higher levels of certainmicro-RNAs (miRNAs), and lower levels of certain other miRNAs, each ofwhich correlated with angiogenic function, than bone marrow-derivedmesenchymal stem cells.

It is known that pro-angiogenic miR-296 regulates angiogenic functionthrough regulating levels of growth factor receptors. For example,miR-296 in endothelial cells contributes significantly to angiogenesisby directly targeting the hepatocyte growth factor-regulated tyrosinekinase substrate (HGS) mRNA, leading to decreased levels of HGS andthereby reducing HGS-mediated degradation of the growth factor receptorsVEGFR2 and PDGFRb. See Würdinger et al., Cancer Cell 14:382-393 (2008).In addition, miR-15b and miR-16 have been shown to control theexpression of VEGF, a key pro-angiogenic factor involved inangiogenesis, and that hypoxia-induced reduction of miR-15b and miR-16contributes to an increase in VEGF, a pro-angiogenic cytokine. SeeKuelbacher et al., Trends in Pharmacological Sciences, 29(1):12-15(2007).

AMDACs were prepared as described in Example 1, above. AMDACs and BM-MSCcells (used as a comparator) were subjected to microRNA (miRNA)preparation using a MIRVANA™ miRNA Isolation Kit (Ambion, Cat#1560).0.5×10⁶ to 1.5×10⁶ cells were disrupted in a denaturing lysis buffer.Next, samples were subjected to acid-phenol+chloroform extraction toisolate RNA highly enriched for small RNA species. 100% ethanol wasadded to bring the samples to 25% ethanol. When this lysate/ethanolmixture was passed through a glass fiber filter, large RNAs wereimmobilized, and small RNA species were collected in the filtrate. Theethanol concentration of the filtrate was then increased to 55%, and themixture was passed through a second glass fiber filter where the smallRNAs became immobilized. This RNA was washed, and eluted in a low ionicstrength solution. The concentration and purity of the recovered smallRNA was determined by measuring its absorbance at 260 and 280 nm.

AMDACs were found to express the following angiogenic miRNA: miR-17-3p,miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, (members of the ofthe angiogenic miRNA cluster 17-92), miR-296, miR-221, miR-222, miR-15b,miR-16. AMDACs were also found to express higher levels of the followingangiogenic miRNA when compared to bone marrow-derived mesenchymal stemcells (BM-MSCs): miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92 (membersof the of the angiogenic miRNA cluster 17-92), miR-296. These resultscorrelate well with the observation that AMDACs express high levels ofVEGFR2/KDR (see above). Conversely, AMDACs were found to express lowerlevels of the following angiogenic miRNA when compared to BM-MSCs:miR-20a, miR-20b, (members of the of the angiogenic miRNA cluster17-92), miR-221, miR-222, miR-15b, miR-16. The reduced expression ofmiR-15b and miR-16 correlated with the higher levels of expression ofVEGF seen in AMDACs.

6.3 Example 3 Functional Characterization of Amnion Derived AdherentCells

This Example demonstrates different characteristics of AMDACs associatedwith angiogenesis and differentiation capability.

6.3.1 HUVEC Tube Formation for Evaluation of Angiogenic Potency ofAmnion Derived Adherent Cells

Human Umbilical Vein Endothelial Cells (HUVEC) were subcultured atpassage 3 or less in EGM-2 medium (Cambrex, East Rutherford, N.J.) for 3days, and harvested at a confluency of approximately 70%-80%. HUVEC werewashed once with basal medium/antibiotics (DMEM/F12 (Gibco)) andresuspended in the same medium at the desired concentration. HUVEC wereused within 1 hour of preparation. Human placental collagen (HPC) wasbrought to a concentration of 1.5 mg/mL in 10 mM HCl (pH 2.25), was“neutralized” with buffer to pH 7.2 and kept on ice until used. The HPCwas combined with the HUVEC suspension at a final cell concentration of4000 cells/μl. The resulting HUVEC/HPC suspension was immediatelypipetted into 96-well plates at 3 μl per well (plate perimeter must bepre-filled with sterile PBS to avoid evaporation, n=5 per condition).HUVEC drops were incubated at 37° C. and 5% CO₂ for 75-90 minuteswithout medium addition to allow for collagen polymerization. Uponcompletion of “dry” incubation, each well was gently filled with 200 μlof conditioned AMDAC medium (n=5 cell lines) or control medium (e.g.,DMEM/F12 as the negative control, and EGM-2 as the positive control) andincubated at 37° C. and 5% CO₂ for 20 hrs. Conditioned medium wasprepared by incubating amnion derived adherent cells at passage 6 ingrowth medium for 4-6 hours; after attachment and spreading, the mediumwas changed to DMEM/F12 for 24 hours. After incubation, the medium wasremoved from the wells without disturbing the HUVEC drops and the wellswere washed once with PBS. The HUVEC drops were then fixed for 10seconds and stained for 1 minute using a Diff-Quik cell staining kit andsubsequently rinsed 3× times with sterile water. The stained drops wereallowed to air dry and images of each well were acquired using the ZeissSteReo Discovery V8 microscope. The images were then analyzed using thecomputer software package, “ImageJ” and/or MatLab. Images were convertedfrom color to 8-bit grayscale images and thresholded to convert to ablack and white image. The image was then analyzed using the particleanalysis features, which provided pixel density data, including count(number of individual particles), total area, average size (ofindividual particles), and area fraction, which equates to the amountendothelial tube formation in the assay.

The conditioned medium exerted an angiogenic effect on endothelialcells, as demonstrated by the induction of proliferation tube formation(see FIG. 4).

6.3.2 HUVEC Migration Assay

This experiment demonstrated the angiogenic capacity of amnion derivedadherent cells. HUVECs were grown to confluence in a fibronectin(FN)-coated 12-well plate and the monolayer was “wounded” with a 1 mLplastic pipette tip to create an acellular line across the well. HUVECmigration was tested by incubating the “wounded” cells with serum-freeconditioned medium (EBM2; Cambrex) obtained from 5 amnion derivedadherent cell lines after 3 days of growth. EBM2 medium without cellswas used as the control. After 15 hours, the cell migration into theacellular area was recorded (n=3) using an inverted microscope. Thepictures were then analyzed using the computer software package,“ImageJ” and/or MatLab. Images were converted from color to 8-bitgrayscale images and thresholded to convert to a black and white image.The image was then analyzed using the particle analysis features, whichprovided pixel density data, including count (number of individualparticles), total area, average size (of individual particles), and areafraction, which equates to the amount endothelial migration in theassay. The degree of cell migration was scored against the size of theinitially recorded wound line and the results were normalized to 1×10⁶cells.

The trophic factors secreted by amnion derived adherent cells exertedangiogenic effects on endothelial cells, as demonstrated by theinduction of cell migration (FIG. 5).

In a separate experiment, HUVECs were grown to sub-confluence in aFN-coated 96-well plate, and induction of proliferation was tested byincubating the cells with serum-free conditioned medium from each of 5amnion derived adherent cell lines (EBM-2 medium, 3 days). EBM-2 mediumwas used as the negative control, and EGM-2 was used as the positivecontrol. After 48 hours, the cell proliferation was scored by DNAcontent measurements using a Promega Cell Titer 96® AZ One Solution CellProliferation Assay (Promega, Madison, Wis.). Error bars denote standarddeviations of analytical replicates (n=3) and the results werenormalized to 1×10⁶ cells.

The trophic factors secreted by amnion derived adherent cells resultedin an increase in DNA concentration, which is indicative of HUVECproliferation. See FIG. 6, where “CM” is conditioned medium.

6.3.3 Acetylated Low Density Lipoprotein (AcLDL) Uptake for Evaluationof Angiogenic Potency of Amnion Derived Adherent Cells

Endothelial cells and microglial cells in culture can be identified bytheir ability to take up fluorescent AcLDL. If the lysine residues ofLDL's apoprotein are acetylated, the LDL complex no longer binds to theLDL receptor, and instead can be taken up by endothelial cells andmacrophages in a highly cell specific manner.

Amnion derived adherent cells were grown either in growth medium withoutVEGF, or in EGM2-MV (Cambrex) with VEGF, to evaluate the angiogenicpotency of amnion derived adherent cells in general, as well as theeffect of VEGF on the differentiation potential of amnion derivedadherent cells. The cells were cultured in their respective media in12-well plates for 4 to 7 days until they reached 70-80% confluence andsubsequently were incubated with a 10 μg/mL acetylated LDL (Invitrogen)overnight. The cells were then counterstained with Calcein AM(Invitrogen) and evaluated for their acetylated LDL uptake usingfluorescence microscopy. HUVECs as control cells for acetylated LDLuptake were grown in EGM2-MV and analyzed as described above. Amnionderived adherent cells displayed minimal uptake of acetylated LDL undernormal growth conditions, but were induced/differentiated to increasetheir uptake through stimulation with VEGF. See FIG. 7.

6.3.4 Tube Formation for Evaluation of Angiogenic Potency of AmnionDerived Adherent Cells

Amnion derived adherent cells were grown either in growth medium withoutVEGF or EGM2-MV with VEGF to evaluate the angiogenic potency of thecells in general, as well as the effect of VEGF on the differentiationpotential of the cells. HUVECs, as control cells for tube formation,were grown in EGM2-MV. The cells were cultured in the respective mediafor 4 to 7 days until they reached 70-80% confluence. Cold (4° C.)MATRIGEL™ solution (50 μL; BD Biosciences) was dispensed into wells of a12-well plate and the plate was incubated for 60 min at 37° C. to allowthe solution to gel. The AMDACs and HUVEC cells were trypsinized,resuspended in the appropriate media (with and without VEGF) and 100 μlof diluted cells (1 to 3×10⁴ cells) were added to each of theMATRIGEL™-containing wells. The cells on the polymerized MATRIGEL™, inthe presence or absence of 0.5 to 100 ng VEGF, were placed for 4 to 24hours in a 5% CO₂ incubator at 37° C. After incubation the cells wereevaluated for signs of tube formation using standard light microscopy.

Amnion derived adherent cells displayed minimal tube formation in theabsence of VEGF, but were induced/differentiated to form tube-likestructures through stimulation with VEGF. See FIG. 8.

6.3.5 Hypoxia Responsiveness for Evaluation of Angiogenic Potency ofAmnion Derived Adherent Cells

To evaluate the angiogenic functionality of endothelial cells and/orendothelial progenitors, cells can be assessed in regard to theircapability to secrete angiogenic growth factors under hypoxic andnormoxic conditions. Culture under hypoxic conditions usually induces anincreased secretion of angiogenic growth factors by either endothelialcells or endothelial progenitor cells, which can be measured in theconditioned media. Amnion derived adherent cells were plated at equalcell numbers in their standard growth medium and grown to approximately70-80% confluence. Subsequently, the cells were switched to serum-freemedium (EBM-2) and incubated under normoxic (21% O₂) or hypoxic (1% O₂)conditions for 48 h. The conditioned media were collected and thesecretion of angiogenic growth factors was analyzed using commerciallyavailable ELISA kits from R&D Systems. The ELISA assays were performedaccording to manufacturer's instructions and the amount of therespective angiogenic growth factors (VEGF and IL-8) in the conditionedmedia was normalized to 1×10⁶ cells.

Amnion derived adherent cells displayed elevated secretion of variousangiogenic growth factors under hypoxic conditions. See FIG. 9.

In a separate experiment, AMDACs were plated at equal cell numbers instandard growth medium and grown to approximately 70-80% confluence.Subsequently, the cells were switched to serum-free medium (EBM-2) andincubated under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 48h. The cells were subjected to flowcytometric analyses for the cellularmarker CD202b (also known as Tie2, Tek, or angiopoietin-1 receptor), areceptor involved in vascular development and angiogenesis. Conditionedmedia were collected, and the secretion of angiogenic growth factors wasanalyzed using commercially available ELISA kits (R&D Systems). Flowcytometric analyses were performed according as described above, and theELISA assays were performed according to manufacturer's instructions.The amount of the respective angiogenic growth factors in theconditioned media was normalized to 1×10⁶ cells. AMDACs displayedelevated expression of CD202b under hypoxic conditions, as compared tonormoxic conditions. See FIG. 10.

6.3.6 Cardiomyogenic Differentiation for Evaluation of AngiogenicPotency of Amnion Derived Adherent Cells

To induce the differentiation of progenitor cells towards acardiomyocyte lineage, a combination of hanging drop culture (HD, tohalt proliferation of the cells and start the differentiation process)with subsequent treatment of the growth-arrested cells with specificfactors was performed in several stages. Following hanging drop culture,the cells were induced with combinations of activin A, bonemorphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF,also known as FGF2), vascular endothelial growth factor (VEGF, alsoknown as VEGFA) and dickkopf homolog 1 (DKK1) over a period of 16 days.In brief, amnion derived adherent cells were grown to approximately 70%confluence in standard growth medium. The cells were then trypsinized,and washed in buffer as previously described. Drops of 20 μl containing700 cells were suspended in the relevant medium and placed on the innerside of the lid of a 100 mm Petri dish using a multi-channel pipette.The lid was carefully inverted and placed on the top of the dish, whichcontained 25 mL of sterile PBS to keep the drops from drying out. Thehanging drop cultures were incubated for 48 h at 37° C. in a 5% CO₂incubator. Subsequently, the aggregate bodies of the cells werere-seeded into culture plates coated with 0.1% gelatin containinglineage specific differentiation medium for further induction.

Stimulation steps proceeded as follows: Stage 1, 4 days BMP4 (0.5ng/mL); Stage 2, 5 days BMP4 (10 ng/mL), bFGF (5 ng/mL), Activin A (3ng/mL); Stage 3, 3 days VEGF (10 ng/mL), DKK1 (150 ng/mL); Stage 4, 4days VEGF (10 ng/mL), DKK1 (150 ng/mL), bFGF (5 g/mL) (+/−5-10 nM5-aza-cytidine). Subsequently, total RNA of the treated cells wasprepared and qRT-PCR analyses for cardiomyogenic markers were performedas previously described.

The results showed that amnion derived adherent cells can beinduced/differentiated to express various cardiomyocytic markers. SeeFIG. 11.

6.3.7 HUVEC Response to AMDAC-Conditioned Medium

AMDACs were cultured for 48 hours in growth medium containing 60%DMEM-LG (Gibco); 40% MCBD-201 (Sigma); 2% FBS (Hyclone Labs), 1×insulin-transferrin-selenium (ITS); 10 ng/mL linoleic acid-bovine serumalbumin (LA-BSA); 1 n-dexamethasone (Sigma); 100 μM ascorbic acid2-phosphate (Sigma); 10 ng/mL epidermal growth factor (R & D Systems);and 10 ng/mL platelet-derived growth factor (PDGF-BB) (R & D Systems),and then cultured for an additional 48 hrs in serum-free media.Conditioned medium from AMDAC culture was collected and used tostimulate serum-starved HUVECs for 5, 15, and 30 minutes. The HUVECswere subsequently lysed and stained with a BD™ CBA (Cytometric BeadAssay) Cell Signaling Flex Kit (BD Biosciences) for phosphoproteinsknown to play a role in angiogenic pathway signaling. AMDACs were foundto be strong activators of AKT-1 (which inhibits apoptotic processes),AKT-2 (which is an important signaling protein in the insulin signalingpathway, and ERK ½ cell proliferation pathways in HUVECs. These resultsfurther demonstrate the angiogenic capability of AMDACs.

6.4 Example 4 Induction of Angiogenesis by AMDACs

This Example demonstrates that AMDACs promote angiogenesis in an in vivoassay using chick chorioallantoic membrane (CAM).

Two separate CAM assays were conducted. In the first CAM assay, intactcell pellets from different preparations of AMDACs were evaluated. Inthe second CAM assay, supernatants of different AMDACs preparations wereevaluated. Fibroblast growth factor (bFGF) was used as a positivecontrol, and MDA-MB-231 human breast cancer cells as a reference(negative control). The endpoint of the study was to determine the bloodvessel densities of all treatment and control groups.

6.4.1 CAM Assay Using Cells

Three AMDACs cell preparations, referred to herein at Lot 1, Lot 2 andLot 3, prepared as described above and cryopreserved, were used. AMDACswere thawed for dosing and the number of cells dosed on the CAM wasdetermined.

Study Design: The study included 7 groups with 10 embryos in each group.The design of the study is described in Table 6.

TABLE 6 Study groups, chick chorioallantoic membrane angiogenesis assay.Group # of No. Embryos Treatment End Point 1 10 Vehicle control (40 μlof PBS/ Blood vessel MATRIGEL ™ mixture, 1:1 density score by volume) 210 Positive control, treated with Same as group 1 bFGF (100 ng/CAM in 40μl of DMEM/MATRIGEL ™ mixture, 1:1) 3 10 Medium control (40 μl of Sameas group 1 DMEM) 4 10 AMDACS, Lot 1 Same as group 1 5 10 AMDACS, Lot 2Same as group 1 6 10 AMDACS, Lot 3 Same as group 1 7 10 MDA-MB-231 cellsP34, Lot Same as group 1 No. 092608

CAM Assay Procedure: Fresh fertile eggs were incubated for 3 days in astandard egg incubator at 37° C. for 3 days. On Day 3, eggs were crackedunder sterile conditions and embryos were placed into twenty 100 mmplastic plates and cultivated at 37° C. in an embryo incubator with awater reservoir on the bottom shelf Air was continuously bubbled intothe water reservoir using a small pump so that the humidity in theincubator was kept constant. On Day 6, a sterile silicon “O” ring wasplaced on each CAM, and then AMDACs at a density of 7.69×10⁵ cells/40 μLof medium/MATRIGELTm mixture (1:1) were delivered into each “0” ring ina sterile hood. Tables 2A and 2B represent the number of cells used andthe amount of medium added to each cell preparation for dosing. Vehiclecontrol embryos received 40 μL of vehicle (PBS/MATRIGEL™, 1:1), positivecontrols received 100 ng/ml bFGF in 40 μl of DMEM medium/MATRIGELTmmixture (1:1), and medium controls received 40 μl of DMEM medium alone.Embryos were returned to the incubator after each dosing was completed.On Day 8, embryos were removed from the incubator and kept at roomtemperature while blood vessel density was determined under each “0”ring using an image capturing system at a magnification of 100×.

Blood vessel density was measured by an angiogenesis scoring system thatused arithmetic numbers 0 to 5, or exponential numbers 1 to 32, toindicate the number of blood vessels present at the treatment sites onthe CAM. Higher scoring numbers represented higher vessel density, while0 represented no angiogenesis. The percent of inhibition at each dosingsite was calculated using the score recorded for that site divided bythe mean score obtained from control samples for each individualexperiment. The percent of inhibition for each dose of a given compoundwas calculated by pooling all results obtained for that dose from 8-10embryos.

TABLE 7 Amount of medium added to each cell preparation fornormalization of the final cell suspension for dosing NormalizationPellet with DMEM and Final Volume of Cell Line size MATRIGEL ™ CellSuspension AMDACs Lot 1 260 μL 0 μL + 260 μL 520 μL MATRIGEL ™ AMDACsLot 2 170 μL 90 μL + 260 μL 520 μL MATRIGEL ™ AMDACs Lot 3 170 μL 90μL + 260 μL 520 μL MATRIGEL ™ MDA-MB-231  40 μL 220 μL + 260 μL 520 μLMATRIGEL ™All cells used at passage 6.

Results

The results of blood vessel density scores are presented in FIG. 12. Theresults clearly indicate that the blood vessel density scores of chickchorioallantoic membranes treated with each of the stem cellsuspensions, or 100 ng/mL of bFGF, or MDAMB231 breast cancer cellsuspensions were statistically significantly higher compared to those ofthe vehicle control CAMs (P<0.001, Student's “t” test). The medium usedfor culturing the stem cells did not have any effect on the blood vesseldensity. Further, the induction of blood vessel density of AMDACspreparations showed some variation, but the variations were notstatistically significant. This concludes that the induction potency ofeach of the 5 stem cell preparations was approximately the same.

6.4.2 CAM Assay Using AMDACs Cell Supernatants

Supernatant samples from MDA-MB-231 cells and from each of the differentstem cell preparations described in the AMDACs CAM assay above were usedin a second CAM assay. As with the AMDACs CAM assay, bFGF and MDA-MB-231cells were used as positive controls.

Study Design: The study included 7 groups with 10 embryos in each group.The design of the study is described in Table 8.

TABLE 8 Study Design - CAM assay using cell supernatants Group # of No.Embryos Treatment End Point 1 10 Vehicle control (40 μl of Blood vesselPBS/MATRIGEL ™ mixture, density score 1:1 by volume) 2 10 Positivecontrol, treated with Same as group 1 bFGF (100 ng/CAM in 40 μl ofDMEM/MATRIGEL ™ mixture, 1:1) 3 10 Medium control (40 μl of Same asgroup 1 DMEM) 4 10 Supernatant of AMDACs Lot 1 Same as group 1 5 10Supernatant of AMDACs Lot 2 Same as group 1 6 10 Supernatant of AMDACsLot 3 Same as group 1 7 10 Supernatant of MDAMB231 Same as group 1 cells(P34)AMDACs cells were used as Passage 6.

CAM Assay Procedure: The assay procedure was the same as described abovein the AMDACs CAM assay. The only difference was that supernatant fromeach stem cell preparation or from MDA-MB-231 cells was used as testmaterial. For dosing, each supernatant was mixed with MATRIGEL™ (1:1 byvolume) and 40 μL of the mixture was dosed to each embryo.

Results

Blood vessel density scores (see FIG. 13) indicate that the induction ofblood vessel formation by the supernatant of each stem cell preparationdiffered. Supernatant samples from the three lots of AMDACs showedsignificant effect on blood vessel induction with P<0.01, P<0.001, andP<0.02 (Student's “t” test) respectively. As expected, positive controlbFGF also showed potent induction of blood vessel formation as seenabove in CAM assay no. 1 (P<0.001, Student's “t” test). However,supernatant from MDA-MB-231 human breast cancer cells did not showsignificant induction on blood vessel formation compared to the vehiclecontrols. As previously shown, culture medium alone did not have anyeffect.

6.5 Example 5 AMDACs Exhibit Neuroprotective Effect

This Example demonstrates that AMDACs have a neuroprotective effect inlow-oxygen and low-glucose conditions using an oxygen-glucosedeprivation (OGD) insult assay, and reduce reactive oxygen species. Assuch, these results indicate that AMDACs would be useful in treatingischemic conditions such as stroke or peripheral vascular disease, andwould protect against reperfusion injuries resulting from ischemicconditions.

Human neurons (ScienCell, catalog #1520) were cultured as permanufacturer's recommendations. Briefly, culture vessels were coatedwith Poly-L-Lysine (2 μg/mL) in sterile distilled water for 1 hour at37° C. The vessel was washed with double distilled H₂O three times.Neuron Medium (ScienCell) was added to vessel and equilibrated to 37° C.in an incubator. Neurons were thawed, and added directly into thevessels without centrifugation. During subsequent culture, medium waschanged the day following culture initiation, and every other daythereafter. The neurons were typically ready for insult by day 4.

OGD medium (Dulbecco's Modified Eagle's Medium-Glucose Free) wasprepared by first warming the medium in a water bath, in part to reducethe solubility of oxygen in the liquid medium. 100% nitrogen was bubbledfor 30 minutes through the medium using a 0.5 μm diffusing stone toremove dissolved oxygen. HEPES buffer was added to a final concentrationof 1 mM. Medium was added directly to the neurons at the end of thesparge. A small sample of the medium was aliquoted for confirmation ofoxygen levels using a dip-type oxygen sensor. Oxygen levels weretypically reduced to 0.9% to about 5.0% oxygen.

A hypoxia chamber was prepared by placing the chamber in an incubator at37° C. for at least 4 hours (overnight preferred) prior to gassing.Medium in the culture vessels was removed and replaced with de-gassedmedium, and the culture vessels were placed in the hypoxia chamber. Thehypoxia chamber was then flushed with 95% N₂/5% CO₂ gas through thesystem at a rate of 20-25 Lpm for at least 5 minutes. The system wasincubated in the incubator at 37° C. for 4 hours, with degassing of thechamber once more after 1 hour.

At the conclusion of the insult procedure, OGD medium was aspirated andwarm medium was added to the neurons. 24-28 hours later, AMDACs andneurons were plated at equal numbers at 100,000 cells each per well of a6-well plate suspended in Neuronal Medium were added to the neurons andco-cultured for 6 days.

Photomicrographs were taken of random fields in a 6-well plate for eachcondition. Cells having a typical neuron morphology were identified, andneurite lengths were recorded. The average length of the neuritespositively correlated to neuronal health, and were longer in co-culturesof neurons and AMDACs, indicating that the AMDACs were protecting thecells from the insult.

Reactive Oxygen Species Assay

AMDACs were determined to express superoxide dismutase, catalase, andheme oxygenase gene during hypoxia. The ability of AMDACs to scavengereactive oxygen species, and to protect cells from such species, wasdetermined in an assay using hydrogen peroxide as a reactive oxygenspecies generator.

Assay Description: Target cells (Astrocytes, ScienCell ResearchLaboratories) were seeded in 96-well black well plates pre-coated withpoly-L-lysine at 6000/cm². The astrocytes are allowed to attachovernight in growth medium at 37° C. with 5% carbon dioxide. Thefollowing day, the culture media was removed and the cells wereincubated with cell permeable dye DCFH-DA (Dichlorofluorescindiacetate), which is a fluorogenic probe. Excess dye was removed bywashing with Dulbecco's Phosphate Buffered Saline or Hank's BufferedSalt Solution. The cells were then insulted with reactive oxygen speciesby addition of 1000 μM hydrogen peroxide for 30-60 minutes. The hydrogenperoxide-containing medium was then removed, and replaced withserum-free, glucose-free growth medium. AMDACs (either cells designatedas Lot 1 or Lot 2), or BM-MSCs, were added at 6000/cm², and the cellswere cultured for another 24 hours. The cells were then read in astandard fluorescence plate reader at 480Ex and 530Em. The reactiveoxygen species content of the medium was directly proportional to thelevels of DCFH-DA in the cell cytosol. The reactive oxygen speciescontent was measured by comparison to pre-determined DCF standard curve.All experiments were done with N=24.

For the assay, 1×DCFH-DA was prepared immediately prior to use bydiluting a 20×DCFH-DA stock solution to 1× in cell culture media withoutfetal bovine serum, and stirring to homogeneity. Hydrogen Peroxide(H₂O₂) dilutions were prepared in DMEM or DPBS as necessary. A standardcurve was prepared as a 1:10 dilution series in concentration range 0 μMto 10 μM by diluting 1 mM DCF standard in cell culture media,transferring 100 μl of DCF standard to a 96 well plate suitable forfluorescent measurement, and adding 100 μl of cell lyses buffer.Fluorescence was read at 480Ex and 530Em.

Results: Both lots of AMDACs used significantly reduced theconcentration of reactive oxygen species in the astrocyte co-cultures.See FIGS. 14A and 14B. In contrast, BM-MSCs failed to significantlyreduce reactive oxygen species in the astrocyte co-cultures.

6.6 Methods of Treatment Using Amnion Derived Adherent Cells

6.6.1 Treatment of Myocardial Infarction

A male individual in his middle '50s presents with chest pain radiatingto the left arm for more than 20 minutes, shortness of breath, nausea,palpitations, sweating. With electrocardiogram results and a rise andfall of blood levels of creatine kinase, a differential diagnosis ofmyocardial infarction (transmural) of the anterior wall of the heart ismade. After stabilization of the individual with nitroglycerin andstreptokinase, the individual is administered 1×10⁸ to 5×10⁸ AMDACs in0.9% saline directly to the affected area using a cardiac syringe withlocal anesthetic. The individual is monitored on an emergency basis forthe next 72 hours. The individual is further monitored over the nextthree months port-treatment by electrocardiogram and/or dyevisualization techniques to assess the extent of revascularization ofthe infarcted area. Therapeutic effectiveness is established ifelectrocardiogram results are discernably closer to normal than beforeadministration of the AMDACs, or if the infarcted area, as visualized,is discernably revascularized.

6.6.2 Treatment of Cardiomyopathy

An individual presents with breathlessness, swelling of the legs andankles, and irregular heartbeats. After excluding other causes, and witha confirmatory electrocardiogram, a diagnosis of cardiomyopathy is made.A sonogram confirms that the individual has congestive cardiomyopathy.The individual is administered 1×10⁸ to 5×10⁸ AMDACs in 0.9% salinedirectly to the cardiac artery using a cardiac syringe with localanesthetic. The individual is monitored over the next three months forchanges in sonogram readings indicating more normal blood flow, and forimprovement in sensation of breathlessness and reduction in swelling ofthe legs and ankles. Therapeutic effectiveness is established for theindividual if any of these sings show improvement during the monitoringperiod.

6.6.3 Treatment of Peripheral Vascular Disease

An individual presents with cold, tingling feet that turn red upondangling, and pain, weakness and tiredness in the legs. After excludingdiabetes, a diagnosis of peripheral artery disease is made. Theindividual is administered individual is administered 1×10⁹ to 5×10⁹AMDACs intravenously in 450 mL 0.9% saline, and is monitored biweeklyfor the next three months. Therapeutic effectiveness is established ifany of the symptoms described above improve during the monitoringperiod.

6.6.4 Treatment of Peripheral Vascular Disease

An individual presents with cold, tingling feet that turn red upondangling, and pain, weakness and tiredness in the legs. After excludingdiabetes, a diagnosis of peripheral artery disease is made. Theindividual is administered individual is administered 1×10⁸ to 5×10⁸AMDACs intramuscularly in 5 mL 0.9% saline, and/or an equivalent amountintravenously or intraarterially, locally between the digits of thefoot, and is monitored biweekly for the next three months. Therapeuticeffectiveness is established if any of the symptoms described aboveimprove during the monitoring period.

6.6.5 Combination Treatment of Peripheral Vascular Disease

An individual presents with cold, tingling feet that turn red upondangling, and pain, weakness and tiredness in the legs. After excludingdiabetes, a diagnosis of peripheral artery disease is made. Theindividual is administered individual is administered 1×10⁹ to 5×10⁹AMDACs intravenously in 450 mL 0.9% saline, and is monitored biweeklyfor the next three months. The individual is also prescribed Cilostazol,100 mg, to be taken twice daily. Therapeutic effectiveness isestablished if any of the symptoms described above improve during themonitoring period.

6.6.6 Combination Therapy of Peripheral Vascular Disease

An individual presents with a cold, tingling right foot that turns redupon dangling, and pain, weakness and tiredness in the right leg. Afterexcluding diabetes, a diagnosis of peripheral artery disease is made.The individual is administered individual undergoes angioplasty, andsurgery to implant a stent in the femoral artery. The individual issubsequently administered 1×10⁹ to 5×10⁹ AMDACs intravenously in 450 mL0.9% saline, and is monitored biweekly for the next three months.Therapeutic effectiveness is established if any of the symptomsdescribed above improve during the monitoring period.

6.6.7 Treatment of Stroke Using AMDACs

A 52 year old male presents with hemiplegia on the left side of thebody, and partial aphasia. A diagnosis of ischemic stoke is made. Afterlocating the area of ischemia using magnetic resonance imaging, theindividual is prepared for surgery to create an opening in the skull onthe affected side. Once the opening is made, 5×10⁷ to 1×10⁸ AMDACs in1-2 mL 0/9% saline solution are administered to the ischemic area. Theindividual is monitored over the next 7-14 days for signs of improvementin any symptom of the stroke, particularly hemiplegia or aphasia.Therapeutic effectiveness is established if any of the symptomsdescribed above improve during the monitoring period.

6.6.8 Treatment of Stroke Using AMDACs

A 52 year old male presents with hemiplegia on the left side of thebody, and partial aphasia. A diagnosis of ischemic stoke is made. Afterlocating the area of ischemia using magnetic resonance imaging, theindividual is prepared for surgery to create an opening in the skull onthe affected side. Once the opening is made, 1×10⁹ to 5×10⁹ AMDACs in450 mL 0/9% saline solution are administered intravenously. Theindividual is monitored over the next 7-14 days for signs of improvementin any symptom of the stroke, particularly hemiplegia or aphasia.Therapeutic effectiveness is established if any of the symptomsdescribed above improve during the monitoring period.

EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1.-38. (canceled)
 39. A method of treating an individual having adisease or disorder of the circulatory system, comprising administeringto said individual a population of cells, wherein at least 50% of thecells in said population are human amnion-derived adherent cells,wherein said amnion derived adherent cells are adherent to tissueculture plastic, and wherein said amnion derived adherent cells areOCT-4⁻ as determinable by RT-PCR, and CD49f⁺, CD90⁺, and HLA-G⁻ asdeterminable by flow cytometry.
 40. The method of claim 39, wherein atleast 70% of the cells in said population are said amnion derivedadherent cells.
 41. The method of claim 39, wherein at least 80% of thecells in said population are said amnion derived adherent cells.
 42. Themethod of claim 39, wherein at least 90% of the cells in said populationare said amnion derived adherent cells.
 43. The method of claim 39,wherein at least 95% of the cells in said population are said amnionderived adherent cells.
 44. The method of claim 39, wherein at least 99%of the cells in said population are said amnion derived adherent cells.45. The method of claim 39, wherein said population of cells isformulated in a hydrogel solution.
 46. The method of claim 39, whereinsaid disease or disorder of the circulatory system is myocardialinfarction.
 47. The method of claim 39, wherein said disease or disorderof the circulatory system is cardiomyopathy.
 48. The method of claim 39,wherein said disease or disorder of the circulatory system is aneurysm,angina, aortic stenosis, aortitis, arrhythmias, arteriosclerosis,arteritis, asymmetric septal hypertrophy (ASH), atherosclerosis, atrialfibrillation and flutter, bacterial endocarditis, Barlow's Syndrome(mitral valve prolapse), bradycardia, Buerger's Disease (thromboangiitisobliterans), cardiomegaly, carditis, carotid artery disease, coarctationof the aorta, congenital heart defects, congestive heart failure,coronary artery disease, Eisenmenger's Syndrome, embolism, endocarditis,erythromelalgia, fibrillation, fibromuscular dysplasia, heart block,heart murmur, hypertension, hypotension, idiopathic infantile arterialcalcification, Kawasaki Disease (mucocutaneous lymph node syndrome,mucocutaneous lymph node disease, infantile polyarteritis), metabolicsyndrome, microvascular angina, myocarditis, paroxysmal atrialtachycardia (PAT), periarteritis nodosa (polyarteritis, polyarteritisnodosa), pericarditis, peripheral vascular disease, critical limbischemia, phlebitis, pulmonary valve stenosis (pulmonic stenosis),Raynaud's Disease, renal artery stenosis, renovascular hypertension,rheumatic heart disease, diabetic vasculopathy, septal defects, silentischemia, syndrome X, tachycardia, Takayasu's Arteritis, Tetralogy ofFallot, transposition of the great vessels, tricuspid atresia, truncusarteriosus, valvular heart disease, varicose ulcers, varicose veins,vasculitis, ventricular septal defect, Wolff-Parkinson-White Syndrome,endocardial cushion defect, acute rheumatic fever, acute rheumaticpericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis,chronic rheumatic heart diseases, diseases of the mitral valve, mitralstenosis, rheumatic mitral insufficiency, diseases of aortic valve,diseases of other endocardial structures, ischemic heart disease (acuteand subacute), angina pectoris, acute pulmonary heart disease, pulmonaryembolism, chronic pulmonary heart disease, kyphoscoliotic heart disease,myocarditis, endocarditis, endomyocardial fibrosis, endocardialfibroelastosis, atrioventricular block, cardiac dysrhythmias, myocardialdegeneration, cerebrovascular disease, a disease of arteries, arteriolesand capillaries, or a disease of veins and lymphatic vessels.
 49. Themethod of claim 39, wherein said population of cells comprisingamnion-derived adherent cells further comprises a second type of cells.50. The method of claim 49, wherein said second type of cells areembryonic stem cells, hematopoietic stem cells, blood cells, stem cellsisolated from blood, placental stem cells, nucleated cells fromplacental perfusate, umbilical cord stem cells, blood-derived nucleatedcells, bone marrow-derived mesenchymal stromal cells, bonemarrow-derived mesenchymal stem cells, bone marrow-derived hematopoieticstem cells, crude bone marrow cells, adult stem cells, orfully-differentiated cells.
 51. The method of claim 39, wherein saidpopulation of cells is administered by bolus injection,intraperitoneally, intraarterially, intramuscularly, intradermally, orsubcutaneously.
 52. The method of claim 39, wherein said population ofcells is administered within an area of ischemia.
 53. The method ofclaim 39, wherein said population of cells is administered peripheral toan ischemia.
 54. The method of claim 46, wherein at least 70%, 80%, 90%,95%, or 99% of the cells in said population are said amnion derivedadherent cells.
 55. The method of claim 47, wherein at least 70%, 80%,90%, 95%, or 99% of the cells in said population are said amnion derivedadherent cells.
 56. The method of claim 48, wherein at least 70%, 80%,90%, 95%, or 99% of the cells in said population are said amnion derivedadherent cells.